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Nasser- Yalpani I .
i
B.Sc., Simon Fraser University, 1977
M.P,M., Simon Fraser University, 1981
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF ppp
- ------------------ --- --
DOCTOR OF
in the ~k~artrnent
Biological Sciences
@ Nasser Yalpani 1987
SIMON FRASER UNIVERSITY
November 1987 q
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P a r t i a l c h a r a c t e r i z a t i o n and p u r i f i c a t i o n o f a g i b b e r e l l l n b i n d i n g p r o t e i n f r a c t i o n from cucumber s e e d l i n g s +ig;
-2 . '?
- Examining Committee:
Chairman: D r . R . C . Brooke
D r . L.M. S r i v a s t a v a , P r o f e s s o r , S e ~ j i o r ,, - S u p e r v i s o r P
. a .
5
7 " d I,
D r . Lawrence ~appapor ' t , /eb(edt . # Vegetab le Crops , U . o f C a l i f o r n i a , Dav is , C a l i f o r n i a
. -- . - D r . G.R.M. Towers, Botany Depar tment , U . B . C . , Vancouver; B. C . , P u b l i c Examiner
t
I , .Y . . I f l - - - , A s s i s t a n t Profe-
i c Examiner
- D r . Bruce A. Bbnner , A s s o y i a t e P r o f e s s 0 7 , Botany D e p t ~ , U . o f C a l i f o r n i a , Dav is , CA, 95616, E x t e r n a l Examiner
I
Date Approved T
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T i t l e of Thesls/ProJect/Extended Essay ,
,Partial characterization and purification of a gihbe~cll i r ~ t ~ i ~ ~ t l i r ~ f r 1 1 r c ~ t c h i 1 1 a
1
Author:- , r.
(s ignature)
->
Nasser Yalpani
(name 1
7 , ) (date) ' -
1 I 1 .,
- - . ,
' A DEAE filter paper assay was, used far the partial I
p r i f ication 0-f gibberellin binding protein (GABP). containing - )
I - extracts from cucumbek seedlinps. The procedures used for this ! - -
, - in vitro assay were,refined to allaw reliable detection o f GABPs --
1 't
with characteristics expected of GA *receptors.
+ 4 L - - - - - -- -
The GA binding.properties-of a ~ y t o s o l fraction -from
hypocotyls wa$ examined using [,H~GA, and over 20 GAS, GA
5q 9' derivatives and oth.er growth substances. The results demonstrate
7 -
structural specif i ~ i ' t y of the binding protein for r-lactohic. 9
C - 1 9 GAS with a 3 0-.hydroxyI-and a C 7 6 carboxyl group.
d7
Additional hydroxylations of the A , C, or D ring of the : \ - B
b
ent-gibberellane skeleton or methylation, of the C - 6 c%rboxyl - imp%de or abolish binding affinity. ~ h e k e - in'vitro results a*
-
generally supported bi & vivo bioassay data. H O W ~ ~ ~ ; , GA: anb - - - - -- -- -- - -- -- -- - - - - - -- - -
GA,,, both considered to be precursorS of the presumably active ,
GA, in cucumber, have low affinl y for the binding protein. With /r / these data inferences about the active site of the.putatnive GA,
receptor in cucumber were made possible. 4
Partial purification of the, GABP fraction was"achieved by \
(NH,),SO, fractionation.and DEAE cellulose chromatography. 1
-/
FurtherLpurification could be achieved using a hydroxylapatite . -
P I
- + or Blue dextran-agarose column, or by pH fractionation.
FPLC-chromatofocusing and -aionexchaige chromatography did not - & result fn enhafced specific binding.
Q *' . - D I
d i i i e
- L - + -
- - -
b
1 I - .- -
li
-- -- TABLE OF , CONTENTS
# , . 1
- i
-- - v - 7 - - - - - .. Approval . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . ~ ~ ~ ~ A A ~ . . . . . . . . . . . . . . i i I . e
- - 'r d ................................................... Abstract i i i +
List of Tables ............... ............................. v i i
.............................................. 'List of Figures ix
Acknowledgments . . . . . : . . . . . . . . . . . . . . . - g o * - ................... xi -%
P * . ...... ............................. A. In<troductibn .L .--. ._. J_ - - - - - -
. . d. Literature Review .................. ..%. ............... :. 5
- - I. Introduction ............................... . " ........ 6 i
I 11. Expected characteristics of GA Receptors 7 - -- --
/
........................ 7 1.- Finite binding capac'ity--.-. 7 . ' P' 2. High affinity and reversibility .....-............. -- 8
% . ....... 3. Hormone spec~ficity and receptor-lability 8 ,
. . 2. Tissue specificity ............................... 9 f 1
5. Correlation with biologicai responke' ............ 1 1 il
- - ,I I,r Pcc-e -p70- - -- - - - ---- - -- - --
? .................................... r ......... ,3V. Evidence for the Existence of GA Receptor? 16 -
t -- - I
V. Conclusions .............. . . . . . . . . . . . . . . . . . . . . . . . . . . 25 a
*
C. - In Vitro Assay -- of - Gibberellin Binding Proteins (GABPS) .. 26 - .............................. I. Materials and methods 28
-
1. Plant material ................................... 28 - . ................ 2. Gibberellins and other compounds 28
3. GLC of GAS ...................................... 33 -
4. HPLC of GAS ....... ; ...................... L . . . 3 f - - - - - - - - - +--
5. GAPB 'extraction .................................. 3 4 -
6. Incubations ..................................... 35 B - + -
*% 8. -- In v-ivo assay . .I.. . c:. ......................... 3 6
-3 -- - - - - - - - -- ---- - - - -- -- 4*
7% -' "/ - 11. Results ...................,...>...................... 38 ' B
1 . DEAE-CelluloseS filter assay ...................... 38
D. characterization of GA Biriding tes :... ................ 53 . .... ........................ I. Materials and Methods 55 - -
5 , L
- ,_1, Extraction of GABPs ....... .-.I. .................. 5 5
.................................... 2.\Gibberellins 55 a .
3. - In vitro binding assays . . . . z . . . . ; . . . . . . . . s . . . . . . 57
.... 11. Results :........;..$..................+......... - - 58 -
3 ......................................... 111. isc cuss ion 65 i
................ E. Partial Purification of a GABP Fraction. 69 k
' 8 I. --Materials amd Methods C' ............................. 71 -"
1 . Chemicals and bateria~s ......................... 71.. w S N . 4 1 : C
2. Buffer ......................................... - 7 1
b ' . * 4. In vitro GAB@ assays ................... I . . , . . . . . . . . 7 5
................ , - - 5. Sodium dodecyl sulfate (,SDS) PAGE 7 6
/
- 11. Results ..:.................. t . . . . .................. 77
? 1 . Source of tissue extract ;...... 77 . ................. .'& * 2 . Effect 6f di'thiothreitol ........................ 81 .
u
3. Ammonium- sulfate precipitation .................. 8t--
......... 4. Open column ion exchange chromatog~aphy - 87 d d
5. Further purification of DE32 eluate ....... ;..... 8 7 - - - - - - -LL-- -
4 ' "
. 11.1. Discussion .........,................:............. 109 % ' , I
I . ~ e v i e w of the Rapid Molecular ~ffec'ts of GAS Using Substrate.~obilization'in Cereal Aleurone as a ....................................... Model System 115
H. APPENDIX 2 ............................................. 122
..... I. Examination of Other - In ~ i t r o Assays for GABP 124 C ................. 1. Sephadex G-25 minicolumn assay 124
* - k ' 0 - ....... ...... . - 3 Nitrocellulose filter assay .,; .-,-. 12-5-- -- , -
Polyethyleneimine-treated glass fiber filter + -
assay ............................................ 127 -
................. 4. Ultrafiltration membrane assay 130 -
, - - -- - - ............. -11. ~ u r t h e r Attempts at GABP Purification 135
..... 1 . Ultrafiltration-.. ..................... L . . . 135 -
- . 7.. Nondissociating PAGE ........................... ' 1 38 *h
4 - * C . I. "eferehces ............................................. 142
L I ST OF 'TABLES s > i r .
Table a page
.-f: -- - - - -- - ------
Cl.:Effect of sample volume on GAI, binding to~--Q&ker,discs . .......... . in absents and presence of G-25 eluate. ; ;. 44'
r - C2. ~om~etitidn for ( 'HIGA, bindingrin incubatfon mixtures ...... containing'column buffer, G-25 eluate or BSA. 45
'i. . - C3._In -- vivo cttcumber bypocotyl' assay using GA,, GA, and . ............................................. GAgME. 47
C4.-Effect of [,H]GA, from 2 sources on filt,er paper .- . - - - - binding in presence and absence' of \G:25 eluate'. .-.-. 4.8 - .- nn .* -as 6 # *
*f u s i n g " ~ ~ ~ ~ filteb presoaked" in GA; .on 3 ~ ] G ~ , binding in the5presence and absence of G-25
JC ............................................ luate. 49 - - -
D l . Relative in vitro affinities of various ligands to ' - - --
[3~]~A, Eiinig protein and their relative in vivo --
-- biological activity i n the cucumber hypocotyl bioaksay. .....:...............,................ . j ~ , . 62
- ' D2. Effect of unlabeled competitors on displacement of . ., ..................... [ 3 ~ ] ~ ~ 4 and.[,^]^^, from GABP. 64
1 . I
El. ['H]GA, binding to G-25 eluate from who e seeds +n germinated for various periods of ti e and from
apical parts of hypocotyls from 7 day old seedlings: ......................................... 78
- - - - - - -- - - -- -- - -- -- -- - -- --
E2. Effect of presence of DTT on GA binding to G-25 eluate. 82
E3. Effect of 10 mM DTT on stability of G-25 eluate stored .. at -70 C for-3 days. ..... L . . . . . . . . . . . . . . . . . . . . . . . . . 83 - -
E4. Binding.of [,H]GA, to cytospl fractions cut with (NH4)2S04. .......................................... 85 - ..
E5:Binding of r3H]~A, to cytosol fractions sequentially ................................ cut with (NH,),so,.
E6- Binding of" ['HIGA, to fractions from a 2.5 x 13.5 cm 87 /
DE32 anion exchange column. .......................... 89 -
i 2
E7. Binding of [,H]GA, to cytosol, G-25 eluate, an& fractions from a 5.0 xQ16.5 cm DE32, and a 2.5 x
- - 8 wAP L<* . ................................FB.--- - - ............ E8.,Purification of GABP from cucumber cytosol. 9 j
vii \ < - .
d - .
-. . . . . . . . . . . . . . . ......... . -~ .. - - -~ ~ - - - - -- - - - - - - - - - - - ~ ~ ~ - . - - - --
w - -
, . - - 4
.. -- -a** I*. "
bA4 Dl L .
LO IX32 eludre and racr i o n ~ ~ o u r i o 'I
and bound by Blue dextran-agarose. ..........*.....a. 98 * t
E 1 3 . p[p33~]G~$r6i53ing toDE32 eluate and iractions.elu>eX from chromatofocusing column. (Mono P). ............ 100 .
- -
E l l , [ 3 ~ ] G ~ , binding t6DE32 eluate qnd fractions exuted * > ...... from ion exchange column ( ~ 8 n o Q) at pH,7.0? 1 0 3
3
E 1 2 . " f 3 ~ ] G ~ , binding to DE32 eluate and fractions eluted b ...... from ion exchange column (~ono Q) at pH 5.9. 104
m
E 1 3 . Effect of pH on preciplitation of GABP in DE32 eluate' '1 ........... . o + from hypocatyls. i...................... 106
? A . -- * &.'& - - - - - - -- - -
, ",
I . precipitation ~~''GABP in DE32 eluate from seeds-at ? ..........>.................... . +. different pH values. 107
Y HI. Assay of GA binding to G-25 eluate using the Sephadex L -. G-25 minicolumn assay. ............................. ,- 126
a - - - - - -- - -
-- ---
R2. Competition for binding of [3H]G~,"to PM20 ultrafiltration membrane discs in absence and
F presence of G-25 eluate. .......................... 134
H3. Binding bf [3H]G~, to DE32 eluate retained b y 2' types C of ultrafiltration cones. ......................... 136
.- H4. Binding of ['HIGA,, to DE32 elukte retained by 10 Kdal - ultrafiltration membranes. ......................... 137
-. -
-- - - H!L ~ , i n d i n ~ i ~-3aGt~ ~ 3 2 .
p , preparative PAGE. 140 \
H6.'Effect od preparative electrop i s buffer on [$H]GA, binding to DE32 eluate. ............................ 141
1. 4
> LIST OF FIGURES A d
Figure - - Page
B1. ~ypothetical sites of binding between a C-19 GA and a specific receptor in the case of optimal spatial correspondence: a) GA, and'the dwarf pea receptor; b) GAT and the cucumber receptor. .................. 10
+ E I . - - B2. ~nalysis of interactions between ~ ~ ~ r n o l e ~ u l e s and GA
binding co.mponents in an idealized system. ......... 15 . . C1 . . Structures* of GA;, GA,, GA,, and GAu methyl ester. ..... 29 .
C2. GLC elOCi0n paof %f 3 3 K ] ~ ~ , - p a r t i a ' ~ ~ p u r i - f - i e $ - f r ~ m - L - - - .............................. NEN reaction -products. 31 -
I - - C3. HPLC-RC elution profiles of [ 3 ~ ] ~ ~ u
-. from NEN reaction products ( 6 . ) Amersham '(b-). 32
- - -
C4. Effect of wash volume on , [ 3 ~ ] G ~ u binding to DEAE filter -
-paper. .....................................'......... 39 C5. Effect of pH on L 3 ~ ] G ~ , binding to DEAE cellulose -,
filter paper. ......................................' 41 - d - - C
C6. Effect of sample voJume on [ 3 ~ ] G ~ , binding to filter paper. .,........................................... 42
Dl. Structures,of G-gibberellane and of ligands used. .... 56 v
D2. Displacement of E 3 H ] ~ ~ , binding from cucumber cytosol by GA, ( a ) and"AA,, compared to GAu (b). ........... 59
D3. ~xchan~eabilit~ of [ 'H]GA, binding to G-25 eluate. ... ;. 60 - -
-El. Scatchard plot of specific binding of GAu to G-25 I d eluate from upgerminated-seeds. ....... =............ 79
B2. Scatchard plot-of specific binding of GA,,to cytosol from apical and basal hypocotyls of 7 day old ~eedli~gs. ....................................;...'.. 80
E3. ~lution profiles of G-25 eluate on 2.5 x 13:5 cm ( a ) % and 5.0 x 16.5 cm ( b ) DE32 anion exchange c?l~umns. . 88
- , - - - - -
F E4. ~Sturability of GA, binding by the HyAp-bound fracthon. 93 -- - - - - - - - - -
_E.5 .. Scatchard ana1ysi.s of bound fraction. i . . - .......... 94 -
2-. ,
Elu t ion p r o f i l e of DE32 e l u a t e ( a ) and HyAp-bound (b) ~ H ~ ~ e ~ e ~ + ~ ~ * * ~ c v r u i ................... : . - .1. 9 5 ,
% ....... . SDS PAGE of G-25 e l u a t e &nd HyAp-bound f r a c t i o n . 96 > .
Elu t ion p r o f i l e of ~ C 3 2 e l u a t e from a chromgtofocusing .. 1, . c
column. r . . . . . . . . . . . . * . * * . . . . , = . . . . . , . ' . ; s . . . . . . . . . . 9..
Chromatography of DE32 e l u a t e on Mono,Q ion exchange ............... column a t p ~ 7 . 0 i a ) and pH 5.9 ( b ) . 1 0
~f f e c t 3 of wash volume on [ 'H IGA, binding to., n i t r o c e l l u l o s e f i l t e r paper. ....... 1. ............. 128 \ - - * E f f e c t of sample volume and presence and absekwe o f 0
G-25 e l u a t = on [ 3 ~ - J G ~ , binding to-nitroceLlulose- - --- ........................................ membranes. 129 #
E f f e c t of 'was& volume on [ ' H I G A , binding t o PEI - t r ea ted g l a s s f i b e r f i l t e r s i n the absenck and presence o f . ....................................... G-25 e l u a t e . 131
-- - -- --
E f f e c t . ~ • ’ - w a s h volume on f 3 H I G A h bindina t o u l t r a k i l t r a t i o n membranes in the pr;sence a n d ............................ absence of G-25 e l u a t e . 1 3 3
,- i wish to thank Dr. L.M. srivastaG for his supervision of *
tvhe research and for his advise during the preparation of this
m h e s i s . My thanks also go t'o the members of my supervisory* -
committee, Drs. L. Rappaport a,nd W.E. Vidaver, and -the other
members of the examining committee, Drs. B.A. Bonner, J.E.
Lister and G.H.N. Tow,ers, for their helpful d-iscussions and- - - - - -
suggestions.
I am grateful to the late Dr. B., Keith whose pioneering work - -
. on gibberellin binding proteins formed the-foundation of this - thesis.
Z.H. Liu helped with the purification-of tritiated
gibberellins. - Numerous - enthusiastic people assisted by 3
harvesting the kilogram quantities of the cucumber hypocotjls --- - - - - -- - - - - - - - - - -- - - - -- -- - -- -
needed for this project. The help of G. Brandson, J. Ch-isholm, s
S. Cheong, C. Dykstra, M. Dykstra, S. MacFarlane, D. ~ c ~ i n n o n , C L
P. Newitt, C. Schnee, C. Srdnanovic, K. ~imewe'll, A. Wong, G.
Wopwitka, Y. Yalpani and R. Zilabi is therefore greatly ,
- To Paula Luciw, my wife, I owe much fpr her patience and
moral support throughout this study. Her encouragements- and
understanding were invgluable.
PART A '
- .
gibberellins ( G A S ) have been recognized to be present-throughout a - - - L p - -- - - 0 .
most of the plant kingdom and-to affect a wide range of >
physiological processes in pfant development (see ~igure 6.4 in P .
Matthysse and-Scott 1984). The most dramatic effect involves the >
promotion of stem growth in genetic or physiological dwarf
plants,. GAS also induce germi~ation \ - of many photodormant seeds,
substrate mobilization in cereal grains, as well as leaf - - -
elongati-on and parthenocarpic fruit set. They also control
flowering and sex expression in- some \gymno- and angiospqrms \ -
-
(~ria; 1966, Jones 1973, Pharis 1985). - - - J
Numerous investigatorsqha%6 exanti-ned the physiological
responses to external GA a~plications or attempted, to identify
the effect of changes in endogenous GA levels. ~huever, the
- sequence of ev<ents involved in linking GAS with their fl
. ,
developmental effects remains largely unresolved. Apy hypothesis - -- I - - & --- - -- - - - --
on the mechanism of GA action has to account for three important -
- - facts. 1 . GAS can af•’ect transcription of genes and translation > --- -
of gene products within several hours of application (Deikman - \ -
b and Jones b86, Hammerton and Ho 1986, Higgins -et al. 1976,
d -- Jacobsen It4undy et al. -- M.undy -- et al.
Muthukrishnan et al. 1983al 2. These effects are observed only -- in response to a few of the more than 70 GAS of plant and fung.ii
-UI
origin (Crosier and Durley 1983). 3. The degree of activity and
of differentiation and .development of the plant tissue
-- and Kazama 19781. f --- - - - - -
he specificity of theL relationpbetween bi.ologica1 activity !
and GA structure as well as the diversity of prkcesses regulated - . $9
by GAS suggest the presence of GA recognition moleculesr '
receptors, that act' as key mediators in GA action in' plants. The P- structural complexity requiredmof such receptors suggests that
they are proteins. A number of studies have b e z undert4k~n t o - -
demonstrate the presence-of and ascribe a functional role t d-r -
such receptor molecules. These studies generally utilized .
techniquespsimilar to those used by endocrinologists studying '
- - - -
the mechanism of action of animal hormones who, in the last 2- b
decades, have clearly shown that receptor proteins pLay a key \ 'role in mediating the effect of steroid and peptide hormones' onc
'
gene expression and on metabolism (Greene and Press 1986, *
Scheidereit -- et al. 1986, Schrader et al. 1981). ~ a s e d upon - - -
- - +v.idenc+ ~o~airna-l-hormom-reci?pto~s; it i s m t s a ~ d t K a t L - - -
plant ,hormone receptors are located on or in responsive cells. &k , . Upon interaction with,.a growth regulator' they become activated, -
9
or combine with it, and then, either alone or in a complex with . - - I
the ligand, exercise metabolic control. - v - -
i Reviews by Kende and Gardner ( 1 9 7 6 ) ~ Rubery ( 1 9 8 ~ ) ~ Stoddart
, (19821, Stoddart and Venis (1980) and Venis ( 1 9 7 7 ) ~ indicated
that there is little experimental support for the existence qf -
- / candidate GA-receptor prot+&s. Hewever, %-R L e e &--
research a reexmination of the published reports 1s appropriate
and will, therefore, he-the focus of a literature review. In --
3 - --
3
evidence in support of the existence of GA d
I
presenked. On this basis experiments were perf'ormed I
I - cucumber seedlings. I
e * - L \ --
PART B
Literature Review
-- - t r i k f t k ~ + + e ~ - ~ ~ ~ - e r - o f - - r e ~ ~ s have
attexpted to'identiiy hnd isolate- the primary Site o GA action I f 4 R
within plants. However, compated to the understanding +ineda - i
about the rnec?hanism o b t ion of auxins ' (Jacobse_n and Hajek --
1987, Libbengar et - al. 1987, Loebler et al. 1987, Veni-s 1987), -- ~rocjress in GA receptor research has been slow. several factors
- - - - - t-
may aecoun for this. GA receptors are expected to be present at 5 low concentrations (picomolar) in cells. Thus purification of
these proteins is a formidable task. Furthermore; in order to be
able to detect such a small number of molecules, radioactive GAS -
oi high purity and specific activity are r'equired. A reliable . source .of--such labeled GAS did not .exist until recently.
0 .z
In this part of the thesis a review of the characteristics
expected of GA receptors and the methods used for theier _ _ - I-,-- -- -- --
m e a s u i n f ill-be followed by an examination of the available 4 + -
literature on GA-binding cell constituents. A- background - 6 .. - I
discussion of a model system of GA action, the cereal aleurone,
is provided in ~ppendix 1 .
1
I f . Expected Characteristics of GA Receptors -
A protein has to fulfill a number of criteria in order to " -
qualifyai a receptor.' These criteria have been discussed in
detail by several- authors (Birnbaumer et al. 1974, Buhlee et al. - - a -- --
--- 1976, Clark and Peck 1980, Kende and Gardner 1976, Smith and - - .. Sestill 1980. Stoddart and Venis 1980) and *ill; therefore; only
1. Finite bindin capacity-. * .
.The -- in vjvo response to'external GA application is satuiable
leg., Brian -- et a). 1964): Howeuer, saturation of.uptake is not > 7
always observed (see Keith -- et al. 1280). Transport, metabolism,,
and/or inactivition by compartmentation by eg., vacuoles
(Garcia-~artinez et al. 1981) change the hormone concentration -- -- - - - - - - - -- - - - - - - --
at the target site. However, since reteptor numbers are limited.
saturable binding*of hormone to-receptor molecules should be
measurable once the impact of these processes has been reduced.
Saturation ic usually demonstrated by exposing the receptor to a rcl-
range of concentrations of radioactively labeled hormone and
. 'subsequently determining the amount of hormone bolnd.
2. High affinity and keversibility 2 I
- - A- -- -- - -- -- - -A
GA concentrations in plant. tissdes are usually very low
(pmol or less than 100 ng g- fresh weight) (Gaskin et ai. 1985, -- Oden -- et al. 1987). GA receptors are, therefore, expected to have
1 4 l
a high-a-ffinity for biologically active GAS. The equilibrium
dissociation constant (K ) of GA receptors is likely to be of d the same order of magnitude as the tissue GA concentration
- > - - - - - - - A PA - - - - --- - - <
to M) (see Clark and Peck 1980, Venis 1985). ,
- Physiological responsiveness to changes in GA concentrations
implies reversible and hence noncovalent Binding of-hormone by @,
the receptor enis is 1985). -
3. Hormone specificitx and - receptor lability B
3 GA receptors are expected to display high specificity for. .J -- - - - - - - - -A - - - -- - - - . ,
GAS, thus enabling them to respond to a ho?monal signal without I P) .
interference from other substances. Among the GAS, v.itr,~ 1
binding af f.inity .should ;oughly correlate with the r&lative P
biologicah wtivity of that molecule. However, correlation mayb 7 * - <
be poor i f facto other than receptor affini$y, such as r . I -
ttans;ort, perm& lity, and perhaps moje importantly metabolic . t
conversions affect~the -- in vivo activity of a substance (eg., Z $ - " Nash it al. 1978). . . - - -
- -- - -- u
On the basis of -- in vivo biodssays and studies,pf GA
metabolism (eg., Brian,et -- al. 1967, Cr zier 1981, Serehryakov et a 0 -
r
important for receptor binding of GAS or GA deri-vatives: a -- - -- -- -- - -
complete gibbane ring system with a 7-laktone ring, a 0-carboxyl
' group at C-6 and a 3 P-hydroxyl group at,C-3. Models of the GA., -
binding sites of the-pea and cucumber receptors were proposed by
., Serebryakov et al. (1984 ) ( ~ i g ; B1). The specificity.of GA -- binding sites can-be anaJyzed by comparing the concentrations of
0 different ligands that-competitively inhibit 50% of the high --
affinity binding of a biologically active, radiolabeled GR.
The observed structural specificity implies that t%e GA -
receptor is a protein. Specific binding is, therefore, e x p e c w
to be diminished by heat and protease treatment. I
* r -. g,
, 4. Tissue specificity
$*
-
The - in-vivo effect'of GAS on plants is influenced by the ----L--L --- - -
type and physiological state of the tissue to which the hormone A
is applied. Thus' in cucumber hypocotyls, GA, more effectively ,
induces elongation in younger than in older tissues (Katsumi and
. Kazama 1978). I n 1 day old lettuce seedlings, maximum hypoco'tyl
growth occurs when GA,'is applied after an initial 4 Qr 8 h of - seedling growth in water. After 24 h little growth promotion is
observed. ( ~ a ~ h n e y and Sr ivastava 1974).
Such differences in tissue sensitivity to GA could result - - - - - -- - - - - - - -
from differences in gene availability and/or receptor numbers, -
There is also the possibility that receptor types differ between . --
Figure B1. Hypothetical szes, of binding betkeen a ~ ' 1 9 GA and a specific -receptor in -the-case of optimal spacial - -- - - - --
correspondence: a) GA, and the dwarf pea receptor; b) GAT and the cucumber -receptor. -The following sites may be conceived on the basis of bioassay data: (1) Site of obligatory binding - a good fit here is necessary for high activity; (XI) sites of ancillary binding - a go@ fit- here increases the activity by some - degree; (111) specific site- of obligatory h drophobic interaction; (IV) site of electrostatic int e ractim between the ionized'carboxyl group on the hormone and a positively b
charged group on the receptor surface. The binding at the alcoholic hydroxyl groups and at the-lactone bridge may be assured either by hydrogen bonds .or by transient formation of an ester linkage. (Copied from Serebryakov et al. 1984, -- with permission from Pergamon Press). The carbon numbers are shown in a ) ,
&"
tissues.' Thus ~ichniewica:and~ana( 1962) rep0rtc.d t b t i - a
, - Myosotis GA3 induces only stem growth while GA, prpmotes stem a
- - - --
growth and flowering. ~ r i a s ,et -- al. (1962) reported that in Grand ' - -
Rapids lettuce the order of effectiveness in ability to promote . <&.
seed germination by G&; > GA, > GA,,, but ability ,to prpmote stem . el~ngation by GAu = GA, > GA3.-It should be noted though that
Crozier et al. -- and Keith et al. 4
-- (1979) observed lettuce
hypocotyl el-mgati-on by_GA7 > GA, > GA,. These experiments - - ----
< ' should be reexamined.
- - + -
5. Correlation with bioloqical response, - -- -
, The demonstration that hormone-receptor binding is
biologically relevant and leads to a hormonal response is
difficult to establish. Comparison of receptor deficient, or I \
enriched single gene mutznts with normal plants can help to
been identified. Ho et al. (1980) screened sodium-azide -- 3
mutagenized barley -for altered sensitivity to GA,. Various GA3 --
insensitive or supersensitive mutants were identified. Some of
these lacked both the GA3-enhanced production,/of a-amylase and
release of phosphatase. The rate of uptake of ['"c-]GA~ in these
( 2 mutants was the same as in the wild-type. The authors suggested - -
that the mutation af fected a regulatory step thas controlled -
both enzymgs. other candidate mutants are the GA insensitive - -- - - - - - - - - - - --
&maize d, (Katsumi -- et al. 1984, Phinney i956), wheat D6899 (Ho et - al. 1981) and pea lk mutants (Reid and Potts 1986). -
0
P -
The measurement 'of recepior-~~ complex format ion is a
- essential for the quantitation and characterization of a = ,
'putative receptor. SueK analysis usually *requires the use of
- radiolabeled hormone of high purity and specific activity \
because receptor numbers are expected to be low.
- + - - - - - - - -
A number of techniques have been developed to separate < -
ligand molecules bound to the receptor from free or unbound ,
k
-
ligand molecules (see reviews by ~irnbaumer 1980, yenis 1985). - -
- -- -- --
Particulate receptor-hormone complexes are readilfi separated --
from free ligand by filtration or centrifugation techniques. -. -
.This is especially suitable for membrane-bound receptors. , ,'- i
Separation of ligand complexed with soluble 'binding sites from -
. . free hormone can be achieved by gel filtration (Schrader 1975)
- or by charcoal adsorption of free liqand (Santi -- et al. 1973).
Protein-hormone complexes may adsorb to ion exchange cellulose~ *
(Santi -- et al. 1973). They may also be precipitated with ammonium
sulfate or trichloroacetic'acid a-nd then washed free of unbound L
hormone (Schrader 1975, Smith and Sestill 1980). Receptor-type
proteins have also been identified anLd quali-t"ified using specific - - antibodies (eg., Dicker-et -- al. 1984), affinity crosslinking
reagents (Jones -- et al. 1984) and autoradiographic techniques
(Young and Kuhar 1979). - - ---- i3.1-----
Ligand - receptor interactions are governed by the
equilibrium:
(Clark and Peek- 1980). If ligand and receptor are at I
equilibrium, then
- (equation I )
where B = mol-of ligand bound, F = mol of free or unbound - - - -- - - -
Z
ligand, n = number of binding sites. Kd = dissociation constant
= I / K , , Ka = association constant.
&I
Estimation of these parameters of ligand-receptor , -- -- -
inte.ractions has to be made atm equilibrium conditi>ons. -
b ~here'fore, assays for ~eceptor-hormone complexes are of limited
use i f thi half-life of such a complex is shorter than the time ,
required for the assay (Stoddart and Venis 1980)-.
Under equilibrium conditions free hormone may bind to two --- - -- -- - -----L-p-- - -- -- --
types of binding sites: a limited number of receptors with high B
affinity and specificity, and a second class, the nonspecific or
background binding sites which have low aff inity but large
. binding capacity (Clark and Peck 1980). To allow.
a characterization of the specific and nonspecific binding
components, total ( = specific + nonspecific) binding is
determined at a range of concenkrations of radioactive hopmone.
- Nonspecific binding is usually meas~~red in parallel incubations
- - --
using the labezed l%gand iTi the presence of a largeexc=ss
(-100X) of unlabeled competitor. As specific binding is,
"
0
,
high affinity and saturable binding sites but does not alter the - - - - - - -+- - ---- --
, - , binding of the radioactive hormone to nonspecific sites, ~f -
-- r specific binding is relatively large compared to nonspecific
binding, sfmple one point assays for receptor binding can be
performed 'by measuring total and nonspecific binding at. a . -
labeled hormone concentration equivalent to the estirna-ted Kd of
the receptor in the presence and absence -of an e x e q s s o f ' + - - -
F, h - unlabeled competitor, respectively. A typical graph expected for
a binding system containing one receptor is shown in Figure -
B2.a.
Am number of matnematica~'transforrnations of equation 1 have
been developed to allow a graphical estimation of those .binding
parameters that govern reeeptor-ligand interactions (e.g.,
Chamness and McGuire 1975, Rosenthal 1967, Scatchard 1 9 4 9 ) . The
most - L - - commonly - - - used - is - - the 'Scatchard plot' using- - - - - - - - -
(equation 2).
By plotting the measured values of B/F versus B, n, Kd or K can a
be estimated, as shown in Figure B2.b. .
Figure B2. Analysis of interactions between GA molecules and GA a
binding components in an idealized system. a) Binding to specific andnonspecific sites. The quantity of specifically bound GA is determined by subtracting nonspecific from total binding. b) Determination of receptor binding parameters using a Scatchard plot. -
IV. Evidence l o r the Existence of GA Receptors
-- -- - -- - - -
Several main approaches have been used in GA receptor
studies. In the fiirst, GA-responsive plants are treated with
radioa'ctive GA -- in vivo. After varying periods of exposure the & .
inter- and intracellular distribution of radioactivity is
. measured by autoradiography or by analysis of tissue extracts
- - 'i
followin,g homogenization and fractionati'on. Receptor-hormone -
. complexes w i ~ h a short half-fiife may not be detected by this
method. Differences in uptake and transport rates,' as well as - metabolism of the applied G A S complicate such analysis of
- receptor, - hormone interactions. The second approach used. in
locating GA receptors involves the in vitrQ measuremedt of the - association of a labeled GA with subcellular fractions after
cell fractionation. This approach assumes-that monitoring GA
act ion is not dep-endent on cellular integrity. Support for such -
approach, identification of those cellular components that are . /
required for GA-induced change% in RNA or protein synthesis is
attempted.
Early support {or the existence of GA recepto;s came from
the work of Johri and Varner ( 1 9 6 8 ) . They demonstrated that . - isolated pea Shoot nuclei shdw quant i tat ive and qualitative
changes in RNA synthesis only i,f GA is present during extraction
and purifi.Ation o f the nuclei. M k a n c o m e n & ~ f - ~ ~ ~ t W s - w r t s - -
greatest when 10 nM GA, was added at the beginning of the-
-
* later steps in nuclear purification. The physiological-ly
inactive GA, had no effect on RNA synthesis. The GA,=enhanced
fraction was not purified and its relevance for & vivo LdC
-
P growth was not investigated. purification of the soluble.f?ctor - -
required in GA stimulation of RNA synthesis does not appear to
, have been accomplished. t \ $ - -
With the objective of finding particulate GA binding sites
in pea stems Ginzburg and Kende ( 1 9 6 8 ) investigated the 7 -
intracellular localization of [ 3 ~ ] ~ ~ , f~llowing~an in vivo 24 h -- b
-
incubation. Microautoradiography showed that label is randomly
distributed throughout the cells. More than 99% of t.he recovered
radioactivity remained in the supernatant after differential
centrifugation of the homogenates. The major part of the
pelleted hormone was associated with intracellular mpmbrane
fractions. This binding to membranes was noncovalent but was not L- - - -- -- -- -- --- -- - - - - --
saturable. Metabolism of the ligand was not exc'luded, nor was
the possibility that exchangeably bound GA, may dissociate from
receptor sites. Abscisic acid (ABA) did not compete for binding.
, In studie%of -- in vivo GA binding Musgrave and coworkers were
unable to provide clear evidence for the presence oi GA.
receptors. Measuring the uptake of radioactive G A S by dwarf'pea
shoots, Musgrave et al., (1969) observed that uptake of L 3 ~ ) G h , - -.
and [3~]GA, by stem sections irfcreases under conditions which - - - - - - - - - -
would result in incre owth promoting activity. Thus \
accumulation of labeled GAS was higher in dark-grown, GA
4
responsive apical stem sections-than in l.iqht-qrown, GA -- <
?sensitive basal shoot- sections. Biologically inactive GA -- -- - - - - - -- - - - - - --
derivative~ did not accumulate as much as active ones. The
uptake of labeled GA was, howevec, not saturable.-In a further
study of GA uptake in barley aleurone layers Musgrave et g . , - - . .-
(1972) noted t.hat the accumu~ation of [ 3 ~ ] ~ ~ , - , [ 3 H ] ~ ~ S , and
[ 3 ~ ] G ~ , methyl ester is not correlated with their biological
activity but with,their degree of-metabolism. While binding was p L L -
not saturable at 2 5 C, the duthors did not examine saturability '
under conditions of inhibited metabolism, s y c h a low
temperature or in the presence of metabolic poisons which in /
bheir study reduced GA uptake. In the absence of metabolism,
equilibrium between receptor and hormone could have become*
established along with receptol saturation. - Asakawa et e., ( 1 9 7 4 ) applied i3H.]GA3 of unspecified purity
and specific activity to bean seedlings over a 2 4 h period to - - - - - - - - - - - - -- -- - -- -- --- - analyze translocabion and intracellular distribution of the
. . hotmo'neb. Plant tissues were then homogenized and fractionated to '
identify the 'location of GA. Most Of the,applied radioactivity
(78.5 to 1 0 8 . 8 ( ! ) % 1 was associated'with thg 1 0 0 , 0 0 0 q
supernatant, very little ( 6 to 9%) with crude nuclear pellets. - -
While failing tb consider metabolism of [IH]GA, or the
possibility of dissociation of the growth regulator from its
binding site, the authors were unable to detect' association of =
radioactivity with pr&eirts, DNA, w - R N A . - - - - - - - - - - -
---- ~si~rg-~o~ague~)us~extract~ion procedures for the cell
r + >
--
fractionation of wheat endosperm, Jelsema et al., ( 1 9 7 7 ) - - ---- -- -- -- - . .
4
detected GA, binding sites in a particulate fraction enriched in . aleurone grains. Binding of GA, was specific and could not be
competed for by GA, but'was by ABA. Specific binding was
suggested to be reversible, with a Kd =- 1.5 pM and 0.45 pmol
binding sites per mg protein. However, in plotting their +
Scatchard diagram the authors used a logarithmic scale for-the-iF
abscissa and thereby overestimated the values of n and Rd by 2
orders of magnitude. No apparent reason was given for choosing I
endosperm as the source-for the extraction. The lack ofl '
extractable specific binding when aqueous procedures were used %
for the of the aleurone grains was also not
explained.
Stoddart et - &. , ( 1 9 7 4 ) incubated excised dwarf pea
. . < - temperature. The 20,000~~ supernatant of homogenates of these "
sections was then examined for GA binding by passing it through
a molecular sieve column and measuring radioactivity in eluate
fractions. i 3 H 1 G ~ , binding by high and intermediate molecular
weight proteins was reversible and pH dependent, the latter
fraction showing highex affinity binding. Equilibrium dialysis
assays confirmed these results and indicated that G k - b i n d i n g is r
specific. [ 3 ~ ] G ~ , was not competed for by the inactive G k , and - - - - --- --- - - - -
3-epi-GA, but was by the growth promoting GA, and 13~]-16-keto
G A I . No saturation of binding was shown. ~etabolism of applied
binding proteins. fie 10,000q supernatant from epicotyl
homogenates was incubated with [ @C]GA, of bnspecified purity.
his extract was passed through molecular sieve and anibn
exchange columns and the radioact.ivity in column eluates
measured. An (NH;) ,SO, assay was also .used. The - - - -
- - ---
results 'obtained failed to indicete receptor-Fype binding.
, Saturation of binding sites.was not shown and is unexpected as
the authors were working with very crude extracts and performed . -* P
binding studies at room temperature, iss so cia ti on of GA from.the . -
binding sites was not taken into account. Furthermore, binding
1 '
was not spg"cif c as GA,/GA, gnd GA,, were equally eff*ective in @ , 8 * ,
displacing about 30% of the bound radioactivity. ~rdtease
>inhibitors were n'ot used although extraction procedures were at
--
room temperature. Thus the risk of receptor degradation was not A -3-- -- --
-- -----pp----- -- ---
In vivo uptake of [,H]GA, by lettuce hypocotyls was observed -- f by Stoddart (1979) to correlate with rates of GA promoted cell *
. elongation. Hypocotyls were incubated at 28 C for 24 to 48 h and
1: bound r-adioactlvity metisured. Of the total absorbed
radioactivity, 95% was associated with the cytosol ,while 4% was
bound by a cell wall containing fraction pelleted at 2,0009. * .
r' This pelleted binding was hypothesized to correlate with GA
- - - - - - -- - -
inducedzchanges in cell wall plasticity. The binding was
observed to be unsaturable, covalent and temperature dependent. - -
-
-
*. - - -
- I Z
AS experiments were.performed at or above 10 C,.Lrnetabolism of - --
- the supplied L 3 ~ 1 G ~ , was observed. - B A
A - - -- - --
On the basis of the GA binding studies discussed to this I
point Kende and Gardner (1976). Venis '(19771, Rubery ( 1981 ) . and
Stoddart and Venis (7980) concluded that there is little 7
evidence in support of the presence of GA receptors. However,
- 't Keith - et * I a1 (1980) reinvestigated this problem with baryey -
aleurone layers. ~ l i ; ~ urable binaing of 'H]GA, to the layersrwhen metabolism of the hormone was stopped with
incubations at 1 - 1.5 C. At temperatures above 3 C metabolism S
occurred and no saturation of binding could be detected: -
.=
Saturating concentrations of GAl but not GAB could displace
bound radioactivi y. t "4
Saturable - in viv.0 binding of GAS to cytosol.of dwarf pea 7
1 5 '
epicotyls was demonstrated by ~ e i th and Srivastava- (,I 980') who*.;
incubations at 0 C to stop metabolism and sliced sections to
reduce transport and permeability effects. Under these
S conditions they were able to detect 2 classes of binding sites
in gel filtration column eluates, one with high'.affinity C K ~ =
60 nM, n = 0.4 prnol.9-I fresh weight). 1 3 H ] G ~ , binding could be * 9..
indibited by. biologic all^ hctjve but not by inactive GAS or r
other'plant hormones. Binding was~susceptible to pro,tease
treatment but was irreversible when assayed in vitro using gel - -- - - - - -- - --- -
giltration columns. Stoddart ( 1982) considered the number of 8
these high affinity binding sites to be too low to be O& - -
-
C
C
- - -
. . - q w - d ~ o g i c a l A g n ~ f ~ c a n c e . Howewer, he failkd to accomt for
loss of binding, sites due to the extraction procedure and pp----Lp-p-pp -- -- ------ ---
I
@ underestimation of binding sites with the nonequilibrium assay.'
*- With an approach similar to that used in pea, Keith et g . , -
< .
( 1 9 8 1 ) were *able t o demonstr,ate - in vitro binding of [ )H)GA, to -
cucumber hypocotyl cytosol usi,ng the gel filtration column
assay. GAQ binding to soluble proteins was shown' to be - . ") - - - - -
-
reversible, saturable and of high affinity. DNA$e, RNAse, - protease ahd heat'trea'tments demonstrated that specific binding. - -
/ "of GA, was to proteins. A good correlation between in vitro -
, -
binding a•’ f inLity and & vivo growth promotion was observed for a ,
ser-ies of GAS. With the development of a very rapid DEAE filter *
paper assay (~eith - et - a1 1 1982), the results obtained with
cucumber could ,be- confirmed and refined. he authors observed a 4
f
-sing.le class of binding sites with a K = 70 nM for GAQ, n = 0.7 . , d
pmop-g-' fresh weight, half-time of dissociation = 6 min for ' , -- -- - -- -- -- --
GA, . Other biologically active GAS compefed for the same binding
sites as [~HIGA, while biologically inactive GAS did not bind.
Specific binding was also detected in GA-nonresponsive basal
sections of hypocotyls or cotyledons. The concentration of
binding sites was the same as in the GA-responsiveapical \
Kypocotyl portions on a dprotein basis. However, on a fresh
, weight basis the apical target tissue had greater amounts of c-
, -
speci.fic binding. Assays using resuspended 130,OOOq pellets and A
' extracts using- hi toTX- rbU supported tKe v i 6 t h a t p s p e c i f ic
binding sites are on soluble proteins.
rn
- Further support for these in vitro res~llts has been rec_ently
provided by studies using etiolated dwarf pea: Lashbrook et-al. -- -- - - -- -- -- - - -- - L
a -- ( 1987) used the Sephadex G-100 assay to demonstrate saturable,
exchangeable - in vitro binding of L3~lGA1 to sn intermediate
. molecular weightAfraction from cytosol. Two pH optima for
. . binding were observed. However, in vitro specificity data were
inconclusive. Liu and Srivastava (1987) ~ s e d the D E ~ f i l t e r - -
paper assay - and - - - 'HIGA. tor khe measurernext of GA binding a - --
proteins (GABPs) in pea cytosol fractions. They observed
saturable, exchangeable binding of [%H]GA, with a Kd near 100 -
nM. There was a correlation b6tween in vivo activity and & -- - -- -
, vitro affinity of the protein for GAu, GA3, and GA, methyl
ester. Scatchard plots suggested similar numbers'of binding
sites in dark-grown tall (cv. Alaska) and dwarf (cv. Progress
No. 9) peas. This should be expected, as these cultivars show
similar growth habits and GA sensitivity in the dark. The dwarf
phe-yp-d-h-fgh Wsemi t i v + t ~ E - wg r e s s No-,!F-are----- 7 expressed ,in light, presumably' due o decreased enzymatic
conversion of GA,, to GA, and possibly also compartmentation of
GA, (Campell and Bonner 1986, Sponsel 1986). he'
Similar efforts by Keith and Rappaport (1987) to show I
receptor-type binding in maize mutants were,-however,
unsuccessful. Although [ 3 ~ ] ~ ~ 1 binding to cytosol was pH L
' sensitive, it was largely unspecific and nonexchangeable at: the
2 pH optima. The effect of pH on 6k stabi 1-iky- (see r-ev-iew 4 y -- --
Takahashi -- et al. (1986) was not taken into consideration. l a c k b
. = +
23
4
. . ', - i
4
I '
-- ofq receptor stability appeared-to have c d i b u t e d to t w e
resul t ' s . .a 4
--
$ .I - - -7-' *
3 . '@
I
0 > -
. -
I t
*
-
- I
P
P
C
-
V. Ceaclusions \
,&
- - --- --
The results from the laboratories of Rappaport and
Srivastava provide strong evidence for the existence of GA
receptors in a number of different plant systems. With the pea B
epicotyl and the cucumber hypocotyl systems many of the criteria -
expecfed of GA receptors are fulfilled. Thus GA binding is
saturable, reversible and - specific with a - - high - - - af - f inityLf_or ' ~--"--)
biologically active GAS, whereas GAS that do not promotesMwth \
do not bind. The develonment of- the in vitro DEAE filter paper -- \ -
assay has led the to allow characterization of GA a - --
structure/activity re1 tionships in the absence of metaboli m - f 7
and permeability can, therefore, determine which
GA(s) among the plant, is actually controlling
development.
The GA binding assay " ; a so permits examination of tizssue
I extracts for GA binding ac ivity and thereby is of great
importance in purif icationl of putative GA receptors,. With a .
I -w
purified receptor the site,(s) of its action and its role in
plant development can be itudied. -
Assay Yitro Gibberellin Binding Proteins
-
- Keith et al. (1982 ) developed the, D U E filter paper assay + - - --
for the quantitation and characterization o f GA--binding protein - -- -- - - -
complex formation. Their work was repeated and some
modifications introduced. These changes and some results
obtained. with other assay methods are described in this chapter :-
- -
/ %
- - -- ,= -
I. Materials ,and methods A
- - --p-pp-pp
1.. Plant material
~ollowing the procedure of Keith -- et a1. (1,982) seeds of
cucumber (Cucumis sativus L. cv. National Pickling, Buckerfields
6 Co., Vancouver, B. C.) were surface sterilized in 50% e
/ . - household bleach, 5 min, and sown in moist, autoclaved
4
(J
vermiculite in flats. Seedlings were grown in darkness at 28
6.5 days, then exposed to fluorescent light,(lO pE-m-*-sec-'
the laboratory for 14 h.
2. Gibberellins -- and other compounds -
f- Structures of the GAS discussed in this chapter are shown in
Figure C 1 . The purity of some of the GAS was estimated by gas -
- - L i qui d e hroma t 09 a p h y t G L C ' t - n & - h-pre s su r e -3 -j;qu-i &-- -
chromatography (HPLC) as described below.
GA, and GA, were purchased from Abbott Laboratories, .> -
Chicago, IL. Their p;rity. was estimated to be > 9 5 % and >go%,-- - b
respectively. GA, (>89% purity) was^ purchased from Sigma. C-7
methyl ester of GA, (GA,ME) was synthesized by Zin Liu-oar me in
our lab by methylation of GA, using ethereal diazomethane and
purified by thin layer chromatography (TLC) to greater than 95%
preparation. [ 3 ~ ] ~ ~ , , prepared by catalytic reduction of GA, and . 1
palladium-catalyzed actions of 3~ gas on GA, was performed by . -- - a - - - - - - - - - - -
NEN, Boston, MA), was purified in our laboratory in cooperation 4
with Zin ~ i u by preparative (Silica Gel G I 1000 urn, Analtech
Inc., Newark, DE) and analytical TLC (Silica Gel 60, 0.25 mm E.
Merck, Darmstadt, F.R.G.). The organic phase of benzene, acetic
acid, water (8;3;5, v/v) mixed with acetic acid ( 1 4 : 1 , v/v) was
- used as the solvent. The purity of the [3H]G~, was estimated by -, - GLC to be greater than 6 5 % , about 5% conkamination was due to
precursor GA, and the rest due to unknown products (Fig. C 2 ) .
The radiochemical purity was estimbted by combined
HPLC-radioactivity counting (HPLC-RC) to be 61% (Fig. C 3 . a ) . The
mass of the [,3~]GA, was estimated by comparing the peak area in - - GLC spectra against those of known concentrations of GA,. The
specific activity of [ 3 ~ ] ~ ~ , was estimated by liquid
scintillation counting of aliquots of , [ 3 ~ ] ~ ~ , of known mass and
- calculated to-bepl.6x100t2 B q mmoT:.' . : - G L C ; M S s p e ~ t r a o f t 5 F - - ~ ~
products purified from model hydrogenation and deuteriation
.reactions of GA, suggested that the final product is indeed
['HIGA, and that the C , ,-C,, methilend g h was not saturated I
(Z . ,H. 'Liu, pers. comm. ) . i
' For some experiments [ 3 ~ ] G ~ 4 with 1 . 4 ~ 1 0 ' ~ Bq mmo17' and
98.1% radiochemical purity (Fig. C 3 b ) was used. This product,
sold by ~mersham Canada ~ t d I ; Oakville, ON, resulted in lower
nonspecific binding ( s e e '~esults' 'l when use& in tve DEAFif-il-fer--
paper assay. It became available during the last stages of this
- R
--
- A - - - - - - - ti
<
0 -
-
1 . .
-
Figure 3C2. GLC elution profile of [ 'H]GA, piptially 'iuiif ied from NEN reaction products. Peaks Pluting at-the retention times of GA, and Gk, are marked. Substances eluting prior to I * ' are contaminants arising from the derivatization compounds used and also appear in control samples containing no GAS. - 4
- - - - - - - -- -- -- ----
> k
, <
- - - - - ,
. &
Figure C3. HPLC-RC elution profiles of [ 3 ~ 1 ~ ~ , partitally purified from NEN reaction products (a) and as supplied by Amersham (b).
-- - - - - - - - - --- --
F
Peak No.
1 2 3 4
\
RT CONC
Pcnk No. R T CONC
1 11.0 0.G 2 k 35.1 0 G 3 37.0 0 8 4 41.5 98.1
-
study only. Most critical experiments were redone with Amersham . F
- t - [ 3 ~ ] ~ ~ , and confirmed the results obt,ained with the product from
--A - - NEN. GAS were stored in ~fhy7acdtate, ethanol 1 I , v/v) at -20
C. All other chemicals used were of analy-tical grade.
Methylated and trimethylsilylated derivatives of the GAS - - -
were prepared using ethereal diazomethane for methylation and
TriSil Z (Pierce Chemical Co,, Rockford, I L ) for
trimethylsilylation according to the method of Binks et al. -- - ( 1969). A Hewlett Packard 5 7 9 0 ~ ga,s liquid chromatograph was
used with a fused silica capillary coluinn (SE-30, 0.2 mm d 12.5 '
\ m, J.&W.--Scientif ic) ifi jection temperature: 260 C, column
temperature: 250 C, detector 'temperature: 28-0 C.
4, E P L C d- f G A S - - &- -------T- - - .- - - - -- - - -
Purity of GAS was also monitored by HPLC. A preparative C-18 \
- rbp reverse phase column (Magnum 9, Partisil 10, ODs 2, ~hatman)'was
used with a Waters gradient chromatography system coupled to a
recorder-integrator. Buffer A, 100% MeOH; buffer B, 1% acetonex
in water adjusted to pH 2.7 with HC1. For.chromatography of GA,
40% buffer A, 60% buffer B was used; for GA,, GA, 60% buffer A,
40% buffer B. Absorbance at 206 nm was monitored. Under these - - - - - - - - - - - - - - - -
condi-tions and a flow rate of 3 ml min- ' , G A , elutes after 22.5 min, GA, after 41.1 min, and GA, after 34.1 min. GA, and.GA,ME
were compared using 85% buffer A , 15% buffer B. Here GA[ and ., a GA.ME elute after 9.7 min and l2:4 min, respectively (see also
- Purity of radiolabeled GAS was measured by HPLC-RC. A' Ranrorta
-
LS flow-through radioactivity monitor (~aytest - - Strahlungsmessgerate GMBH, Straubenhardt, F.R
the HPLC. For analytical runs- split ratio of < - w i t h a
scintillant (Atomlight, NEN) : column eluate ratio of 3 : l was - -
J / - used. Photons emltted from mixed sample and scintillant-in a 2.1
ml measurement cell were measured by photon'detectors. Static
'triti,um counting efficiency was 38% under these conditions. - -
5. GAPB e,xtraction
All pr-ocedures were performed dt 0 to 3 C. Apical 1 .-5 to 2
cm of hypocotyls were'cut and pooled into freshly prepared
phenylmethylsulfonyl fluoride (PMSF), adjusted to pH 7.3 with
H,P04), drained and homogsenized in an equal volume ( 1 : 1 , w/v) of
extraction buffer using mortar and pestle. The homogenate was
passed through 2 layers of cheese cloth and centrifuged at #
100,00Oq, 1.5 h. The supernatant cytosol kas used for some GABP
assays. For most experiments, (NH,),SO, was added to this
supernatant to 60%. After equilibration and centrifugation at e -.
24,000q1 20 min, the pellet obtained was washed in col-umn-
>buffer, particulates removed at 7,0009, 5 min, and the
(~harmacia). The protein fraction eluted was-then used for GABP - - - - - " a
assa-ys and is called (5-25 eluate. Protein measurements were made
according to the method of,Bradford ( i 9 7 6 ) using BioRad protein
assay solu'tion ( ~ i o ~ a d , Richmond, C A ) with BSA as a standard.
6. Incubations
High affinity specific binding of C3~]GA4 by the putative"
receptor has to be dist'ingwished from low affinity, nonspecific
binding by other macromolecules. For this purpose, buffer - -- - -
controls or protein fractions were typically incubated with 50
nM [ 3 ~ ] G ~ 4 in the absence or presence of an excess of selected
unlabeled GAS for 1.5 h. This incubation time was used by Keith
et a1. ; 1981 ) and was also con•’ irrned by my. preliminary -- 4 - t
experiments to be adequate for equil9brat'ion (data not shown).
Metabolism of ligands was inhibited by keeping all solutions at
0 to 2 C (see Keith 9 al. 1980) . -
7. DEAE-Cellulose filter assay
The methodology used is based on that described by Keith et . - a l . (1982)'. At neutral pH the GABP is negatively charged h - 0
binds to D E A E - ~ ~ ~ ~ u ~ o s ~ , filters. Ligands bound by the protein
are retained -- while unbound. molecules_ are washed t h r ~ ~ u ~ h ~ - P r x _ t e - i ~
binding to filter~ is affected by cations, hence low ionic a
strength buffers are used for incubations as well as wash.
35
Stacks of 2 filter discs (2.4 cm diameter, Whatman -DE81) \
' moistened in ice-cold pssay buffer (in early experiments: 10 mM
Tris-K1, T-mPT EXITX rpFI 7.51, later: 10 mM ~-phos~hat~e,' 1 m i
EDTA [pH 7.01) were placed on a vacuum filtration manifold
(Hoefer 225 V). The filters were equilibrated with 25 ml-assay
buffer. The vacuum was released and aliquots of the incubation -
mixture pi'petted onto the filters. After exactly 1 min the
filters were washed with assay buffer to remove unbound ligand - -- - -
molecules. Sample and wash volumes are specified in 'Resultsi.-
The filter stacks were allowed to dry for about 5 sec, placed in
scintillation vials and 1 ml absolute EtOH added. After 30 min 6 - --
ml of scintillant (Scintiverse 2, Fisher) was added, the vials
shaken and left in the dark before counting with a Beckman LS
8000 liquid scintillation counter at about 44% efficiency. The
13H]G~, concentration in the incubation mixture was routinely
determined by measuring th6 radioactivity of 10 ~1 unfiltered
aliquots. Triplicate samples were, us-ed. Sample dev?ation from fl
the mean was-generally less than 10%.
- -
8. In vivo assay --
Cucumbef seedlings were grown in fla9s for 6.5 days in the dark %
as already described. They were then. transferred to a growth
chamber at 2.5 ~ E = r n - ~ * s e c - ' 25 C for 14 h. At this stage the
seedl'ings were about 5 cm tall and were selected for uniformity.
The apical 1 cm of hypocotyls was marked with India . - ink. -- --- GAS --
' were applied-to each hypocotyl in 4 pl of absolute ethanol.
- chamber hypocotyl elongation was measured. Fifteen seedlings
used - - - - - A - -
pgr treatment. were
Only low levels of specific binding were.detected in cytosol
fractions when filter paper assays were performed according to
the procedure of Keith et z&. ( 1982 ) (data not 'shownjf. As the t - - - A A
batch of f -per and ['H~GA, available to me d i f f e r e d L ~ o y
1 that used by those authors i t * was decided to reexamine some c l i _
the basic parameters bf the filter paper assay. Conditions L t ~ r
in=reasing the sensitivity'of GABP detect'ion weri sought. '
, a. Wash volume
The purpose of this experiment was to determine the volume
of assay buffer wash sequired to separate free from bound
[ 3 ~ ] G ~ , on the filter paper. The results shown in Figure C4 - - -- -- - -- - -- -- - - A A - - - - - - -- - - - -- -- - - L- -- - -
indicate that at least 50 ml assay buffer are required t~
k efficiently remove free ['HIGA, from the fi'lt r discs.
'total radioactivity applied to the filter stack, 0.2% is
adsorbed and cannot be washed off (see also Keith -- et al. 1 9 8 2 ) .
A wash volume of 100 ml was chosen to permit small errors in
volume delivery without affecting the extent of removal of
unbound ligand. Exceeding the 100 ml wash or inclusion ~f 0 . 1 M
KC1 in the assay buffer results in a decrease of detectable I
1 3 ~ ] G ~ , binding to the GAEP (data fi'ot shown). This couT8 b~ cK~e --- to increased dissociation of ['HIGA, from the binding siies
and/or desorption of protein fr'om the filter paper during the
A
I
-
-
Figure C4. Effect of wash volume on [ 3 ~ ] G ~ , binding to DEAE filter 'paper. 50 ul >al?iquots of incubation mixture containing 50 nM
s i 3 ~ ] G ~ , were loaded onto filter paper stacks. After 1 min suction was applied and the filters washed with different volumes of assay buffef. Data shown are the average of 2 experiments. Total radioactivity applied = 79,000 cpm : 50 #tl - '3- -
- - - - - - -- -- - - -- - ---
.- -- lengthier wash. The 100 ml wash requires about 1 min, w h i l c t h e L
half-life of"dissociation of GA, from the binding site is less - - - . - - - A - - - - - - - - -- - -
than 10 min (Kejth , - - et al. 19821, andiPar.t D this report). *
' Therefore, hormone binding to protein is not measured at i -
equilibrium and hence specific binding will be underestimated.
b. Sample and assay buffer pH -
The binding of L3HIGA, to the filter paper in the absence of - - - --
cytosol, as well as total binding in its presence, were measured
at incubation and assay buffer pH values ranging from 6.75 to
8.0., Spezific binding was determined by subtracting nonspecific -
-
/
bindi~g, measured in the presence of a 100 fold excess of
, unlabLaled GA,, from total binding. Figure C5 shows that the
radioactivity adsorbed to the filter paper in the presence or
abse~ce of cytosol decreases as pH increases. Small changes'in
pH at pH-7 .5 have d significant effect on total and specific
- a binding.-Moreover,the-di*erence-betweenspecific and- - - - - - ----'
background bjnding is increased below pH 7.0. For these reasons
sample and assay buffers were maintained at pH 7.0 for all
further experiments. L
,
c. Sample volume and protein concentration
s
Figure C6 shows that total and specific binding of [IHIGA,
is linear for sample volumes ranging from 25 to 200 ~1 when a
cytosol preparation containing 2 mg protein*mlb-I is used. -
i3HlG4, binding to a desalted, resuspended 60% (NH,),SO,
precipitate of the cytosol was assayed in the absence and
Figure C5. Effect of pH on [ 3 ~ ' ] ~ ~ , binding to DEAE cellu-lose filter paper. Aliquots of incubation mixture containing sxtraction b"•’fer . , in the presence or absence of cytosol were asgayed at the pH shown. Specific binding was determined by subtracting nonspecific binding from total binding. Cytosol was assayed
' at 1 mgoml-'. . .
- 50 ~MFH]GA~ + cytosol .. =total binding
X-x specifid binding in presence of cytosol
,--4 ~ O " M [ S H ] G A ~ + extraction buffer
~ i g u r e C6. Effect of sample volume on [ 3 ~ ] ~ ~ , binding to E a paper. Cytosol at 2 mg protein-ml-' was used for the
determination of total (0) Bnd specific ( X I b i n d i n g . B i n d i n g absence of cytosol ( * ) was alsormeasured. . in the
i l t e r --
50 '1 00 , 150
Sampb~olume applied to filters (pl)
-- resence 04 an excess of GA r
@ . AS shown in Table C r f , the sample
volume used does not greatly influence results when data are - -
presented on a cpm*ml-' or cpm-mg-I protein basis. This at l'east - .
is valid for samples containing high protein concentrations as ,
found in the G-25 eluate. Table C i also indicates that in the
absence of protein unlabeled GA, can compete for a substantial B
number of the,13HlG~, binding sites on the filter paper. This '
, . 'apparent specific' binding, therefore, contributes to the
P" P
specific binding observed with the cucumber extracts. By loading
> I 0 0 pg protein per sample the proportion of the specific
binding to the receptor is kept in sufficient-xeess over
apparent specific binding to the filter paper.
d. GA binding by nonreceptor components
The results in Table C i indicated that unlabeled GAu
competes for 'binding sites on the ion exchange filter - - - -- - - - - -- - -- - - - - - - - -- - - - - - -L
paper. This, of course, makes detection of GABP containing
fractions difficult. In order to resolve the difference between
'apparent specific' and 'specific' binding a selection of
unlabeled GAS with-different in vivo activity were tested for -- . competition-with [,H]GA,. These were used in incubation mi.xtures
containing column buffer, column buffer + BSA, and G-25 eluate.
GAu with high, GA, with moderate, and GA,ME with little or no i n - vivo activity.were used as competitors. The results i ~ a b l e C2)
indicate that ability for a competing GA to displace
radioactivity from the filter paper correlates with in v i v o
growth promotion only in the cucumber extract. Data on in vivo --
Table C t . Ef feet-of sample volume on GA, binding to f i l t ~ r pi sc+ in absence and presence of G-25 eluate. Resuspended, desalted 60% (NH,),SO, precipitate of cytosol
-- ~ X 3 - ~ ~ o O ~ e i i n . m l - ~ ~ ~ f c~-bllffpr W ~ C irt~ubat-cd for- 1.5 h with 50 nM [3~]GA, in the absence and presence of 5 UM GA,, ~liquots of the incubation mixture were assayed as described in ateri rials and Methods'. Total, nonspecific, and 'specific' 'binding were determined as decribed in Figure . C5. ~ a t a represent cpm bound per sample.
Sample Sample Total Nonspecific 'Specific' - V0L2 (ul) - - - p2
Column 50 390 190 200 buffer 100 1,030 440 590
1
G - 2 5 50 1,000 500 4L
500 eluate 100 7,910 970 -.
940
-
~ 1 - P ? - j r r l r x L. . . --
a A 4 LJll*ll 1'1 l ' l a - - - - -
a L l " ' ' " ' A * L ~ > - L . a .
containing column buffer, G - 2 5 eluatz or.BSA. Data represent cpm bound per 100 ~1 sample.
- - - -- - -- - - 3
,
Incubation in vivo column - G-25a BSA activity buffer eluate
5 0 nM [3HlGAa . 3 8 0 . 1 , 4 5 0 1 4 , 5 8 0 + 5 pM GAa +++ 2 3 0 7 9 0 7 , 7 1 0 + 5 MM GA3 -C + 2 0 0 1 , 0 6 0 1 4 , 6 4 0 + 5 0 pM GAoME 0 2 1 0 , I . , 2 6 0 13 ,860
- - +
a ~ - 2 5 eluate was assayed a t 4 .2 and BSA at 2.8 mg protein-rnl '
biological activi:ies of these GAS and GA derivatives are shown
in able C3. he data from these two tables reinforce the - - - - - - - - - - - - - - - - - -
-
notion, therefore, that detection of GABP containing fractions .. - - rgquires the use of a number of GAS in binding assays in order
to discriminate against nonreceptor GA-binding sites.
The possibility that some of the radioactivity adsorbed by
the filter paper and protein is not but results from - --
- unspecified contaminants pre2sent in the [ 'H~GA, purified in our
laboratory to 61% radiochemical purity was also investigated. P
For-tTls [~H]GA, supplied by Amersham with 98 .1%
radiochemical purity (Fig. ~ 2 ) and a specific activity of
1.4x1012 Bq-mmol-I was used. The results shown in Table C4 show *
that less radioactivity is bound from samples containing' '4
Amersham's ['H]GA, and that the inactive GA4ME displaces less
radiolabel from binding sites in G-25,eluate compared to samples
containing the [ 3 ~ ] ~ ~ , purified in our laboratory. The lower - - - -
- - - --- - --- - - - --
background radioactivity does improve binding data. The Amersham
product became available only in the last stages of this study. a
With it the general trends observed with the product from NEN'
could be confirmed. binding studies.
The results in Tables C 1 and C2 demonstrated that ['HJGA, is
bound by DEAE filter paper and that this binding is, competed -for
by unlabeled GAS.. I t was attempted to reduce this adsorption of
GAS by soaking the DEAE filters in assay buffer containing 10 uM ,
GA, and thereby saturate.al1.. binding sites. Table C5 shows that
this treatment had an insignificant effect and did not improve
Table C3. In vivo cucurnbe; hypocotyl assay using GA., GA, and -
Test compounds were applied in 4 ccl 8 0 % ethanol. The length --
measured after 4 days. -- - Dosages with different - superscripts are significantly different at the 95% confidence level (Duncan's multiple range test) ( ~ u r i and Mullen 1980).
Treatment Hypoctyl Length (cm)
Control l . f a 1 ccg GA, l.9b
- - --
1 ~9 GA3 . 1 . 3C 1 ~g GAQME 1 . l a -
10 pg GA4ME 1 .2a1c t o o pg GA,ME ' 1.2a,c
T a b l e .C4. E f f e c t o f [ 'HIGA, f r o m 2 s o u r c e s on f i l t e r paper b i n d i n g i n p r e s e n c e a n d a b s e n c e o f G - 2 5 . e l u a t e .
- - - - % t a , r ~p~*e~-t-e-pwbmm%-pe~- +W p% ~ m p ~ u a ~ w a - s - - - - - a s s a y e d a t 2 .5 mg p r o t e i n - r n l - .
1
*
I n d u b a t i o n Column G-25 t b u f f e r e l u a t e
i
5 0 n M [ ) H ] G A , ~ 760 2 , 4 9 0 + 5 WM G A 4 4 10 1 , 6 8 0 + 5 f l G A 3 3 6 0 2 , 0 7 0 - - ,-- - -
+ ' 5 0 KM GA4ME 360 2 , 1 0 0 .I
t
a [ 3 ~ ] ~ ~ , p r e p a r e d b y NEN a n d p a r t i a l l y p u r i f i e d i n o u r l a b o r a t o r y
b[ ' H ~ G A , s u p p l i e d b y Amersham i n I986
2 & k + C ' 5 . E f f e c t of using DEAE f i l t ~ r s p r ~ s e m k d i n GA, 0 1 1
[ 3 ~ ] ~ ~ , binding i n the presence and absen'ce of G-25 e l u a t e . Samples were loaded on DEAE f i l t e r s t h a t had been soaked i n assay b u f f e t t n L L t h e - a b E L presence o t '10 ccM unlabeled G A , . Data represent cpm bound per 100, ~1 sample.
Sample -2 column b u f f e r G-25 e l u a t e ' c o n t r o l GA,-soaked c o n t r o l GA,-soaked
50 nM [ 3 ~ ] G ~ , + 5 ccM GA, + 5 BM GA3 + 5 0 1M GA4ME
the quality of the data when NEN [ 3 ~ ] G A , was used, Similar -- -
results were obtained with Amersham [ a ~ ] ~ ~ , . Although - c - - - - - - --- --
radioactivity bound to the filte; paper was close to background.
The basis for these results is not apparent.
The interactiqn between GA,, GABP-and other macromolecule$ is /- '
-
complex. In order to distinguish binding sites oc t h e p e p t o r
from others; a rapid and reliable assay system is required.
Keith -- et al. ( 1 9 8 2 ) had introduced ,the DEAE-cellulose filter - -*- - paper assay for GABPs. That method was showh to be more
efficient than the Sephadex G-50 column assay (Keith et a l . - - - 1981) . Among the methods I tested (see APPENDIX 2 ) , the DEAE
filter assay was also shown to be the method of cQoice.
-
On the basis of my experiments some minor changes to the
procedue of Keith et al. f 1 9 8 2 ) were introduced to enhance the -- difference between measured ' specific' and 'nonspeci •’ ic'
binding. Thus the volume of assay buffer, used to remove the
free ligand from the filter paper, was increased from 75 ml to -
100 ml - and its pH changed - - - -- - from - - - pH ,5 t o 7,J, - - -- -- -- --- *
B
While these changes result in some improvement of data I
quality they also have some tdrawbacks. Thus by increasing the
wash voiume the likelikiood of GABP being washed off the filt-er t
paper and the possibility of increased GA -, GABP dissociation
enhanced. Furthermore, Keith et al. ( 1 9 8 1 ) have shown that --
binding of [ 3 ~ ] ~ ~ , to the GABP has an optimum at pH 7.5 when
assayed using Sephadex G-50' columns. In that assay system
separation of bound from free ligand is not likely greatly
affected by sample pH. Wiih the DEAE filter assay used here d
small changes in pH near pH 7.5 appear to have a4igni f icant
of 7.0 was chosen for the DEBE filter assay because the 1
- - - - - - - - - - -
difference between specific and background bindinq is enhanced .
and small changes in pH have little effect on K3H]GA, bound
(Figs. C 4 , C5). The choice of pH and wash volume will,
therefore, result in an underestimation of the number of
receptoc binding sites. Further limitations to the assay are
imposed by the purity of the radiolabeled GA,\as evidenced by
the comparison -of the partiall) purified NEN [ 3 ~ ] ~ ~ , with the
Amersham product able C4).
Displacement of :[3H]GA, by GA, occurs not only from the
binding site of GA recep t -o r - l ike 'p ro te ins , but also the DEAE
filter paper and nonreceptor proteins such as BSA able ~ 2 ) .
These nonreceptor entities contribute to 'apparent specific' . D
binding. Receptor-type specific binding therefore can not be
presence of an excess of 'unlabeled GA, from total binding. In
\L order to screen tissue extracts for the presence of
receptor-type proteins a more .rigorous assay involving a series
of biologicalty act<vepaila inactive c~mpet2to;s is required.
Such assays may not be able to discriminate between [ 3 ~ ] ~ ~ ,
binding to receptor-like proteins and binding to enzymes of GA
biosynthesis or metabolism (eg., hydroxylases or glycosylases).
PART D '
characterization of GA Binding Sites
t
-'2
3 C when metabolism of GAS is alhost nonexistent (Keith et -ale - - * - - -- -- - --
1980). The possibilitjes of conversion of the GA or GA
derivative supplied to a different ftrm during the assay are - 2
therefore negligible, Furthermore, since the assays are
performed - in vitro using'cytosol, the concentration of the GAS /-
used reflect the concentration at the binding sites. The filter,
assay phus allows a study of the affinity of the GA binding ' - -
sites for different GAS.
' ~ e i t h et al., (1981, 1982) have shown that the GABPs in -- 1
cucumber cytosol bind GA, with high affinity ( K ~ = 70 nM). This
binding is saturable ( n = 0.4 pmol-mg-'- soluble protein) and
exchangeable (half-l2fe of dissociation = 6 to 7 mi'n). Moreover,
by double reciprocal plots they shoved that GA, competes for the
same binding sites as GA, but the inactive GA,, does not.
- ----- -- In this chapter I shaflpreFrt-on the binding of a series of
biologically active and inactive GAS, GA d&rivatives, and other 4
plant growth substances to a partially purified cytosol
preparation. The res6lts provide information on t h o ~ e features
of the GA molecule that are impgrtant' in GA--receptor protein
interaction.
-
1.Extraction - of GABPs
G-25 Eluate was prepared as
Assay of GABPs'.
described in Part C 'In Vitro
Structures of the GAS and other compounds discussed in this
chapter are shown in Figure Dl. i3HIG~, had been synthesized-by
NEN and was partially purified in our laboratory as desc.ribed i
Part C . i 3 ~ ] G ~ , , prepared by catalytic reduction of GA,, was
also used in some experiments. Its purity and specific a c t i v i t y
were similar to that of the NEN [ 3 ~ ] ~ ~ , . 3-I3pi-G~. and C - 7
methyl esters of GA, , GA4 ,+and GA, were s y n t h e s i zed and+r - iLkL --
by TLC. GA, and GA, were purchased from Abbott ~aboratories,
Chicago, IL; GA, was a gift from Dr. G. Sernbdner, Institut f u r
Biochemie der Pflanzen, Akademie der Wissenschaften, Saale,
G.D.R.; 2,2 dirnethyl GA. (DiMeGA,) was a gifT from D r . H.
Pharis, Dept. of Biology, University of Calgary, Alberta. The
other GAS were generously donated by Dr. L. Rappaport, Plant
Growth Laboratory, Dept. of Vegetable Crops, University cf
California, Davis. All GAS were stored in ethyl acetate, e t h a n C 1 - ---
( 1 : 1 , v / v ) at below -20 C. Some of the rare GAS were available
in only minute quantities. Their purity and mass could n o t be c
a Figure D l . Structures of ent-gibberellane and -
]adapted from Bearder 198p) of ligands used.
verified in our laboratory. Some of these were assayed only
repeat experimen S . 4
3. In vitro binding assays "
* --
DEAE cell'ulose filter assays for determination of bound
radioactivity were performed as described in ~edtion C, In ~ i t r o - Assay of GABPs.'
Estimates of the affinity of the GABPs 'for the selection of. i
different GAS and other substances were obtained by measuring
the amount of [ 3 ~ ] ~ ~ , binding displaceable by various
concentrations of unlabeled competitor. In a typical experiment
G-25 eluate was incubated with 50 nM I3HIGA, in the absence or b
presence of competing ligand concentrations radging from 50 nM
&. [)H]GA, of up to 10,000 times. In so-me instances, the competitor
concentration ranged to 0.25 mM. In each experiment competitors
were compared to unlabeled GA, as a standard. 100 ~1 samples
containing 350 to 390 pg protein were used in the binding assay.
Two or 3 replicates were used for each sample.
11. Results ' pp - -- --
0
- - - - -
* The affinity of the bindin^ in^ protein for various ligands . '
was .evaluated by studying the influence of their increasing * b
concentration on the binding of E3H]GA,. Figure D2a illustrates
the competition curves obtained using unlabeled GAu and GA, as
competitors. Total binding is the.amount of [ 3 ~ ] G ~ , bound in the
absence of any competitor. Nonspecific binding represents the -. LA
0 amount of radioactivity bound in the presence of an e x c e s s of 9
competitive ligand. Specific binding is the difference between 8
total and nonspecific binding. It is noncovalent and 1
exchangeable (see also Fig. D3). As the concentration of B
unlabeled ligand in the incubation mixture is increased, the 1
number of exchangeably bound i 3 H I ~ A 4 molecules is proqressively
reduced and the radioactivity adsorbed to the filter paper a
decreases. At 5 pM GAu the competition curve forms a plateau. At
&his poirrt a1 1 those bindi-pig-psi terwfric-fi reversibly % i ~ & t ~ f 5 l C ~ , '4
- are saturated by the excess of, unlabeled GA,. The amount of
radioactivity still bound results from sites which b i ~ d PH~GA,
irreversibly or with low affinity and is, therefore, nonspecific
binding. The GA, dispiacement curve reaches the same plateau as \
that of GA,. However, a much highe concentratiop of G k , 1s - i
required to saturate all -specific binding sites. The tlnding
protein, therefore, has a lower affinity for GA, than for Gk,. . As a measure of the af f i n i t y ~ f t#e bindiftg p r o k e i n f o ~ - a - A-
ligand the I,, vblue for that ligacd was determined. I,, is the . a
1 Figure D2. Displacement of [ 3 ~ ] ~ ~ , binding from cucumber cytosol *by GA3 (a) and4 GA,, compared to GA, fib).
A 60% (qH,),SO, pellet of cucumber hypocotyl cytosol was desalted. This fraction containing 3.6 mg soluble protein
, ml-' was incubated with 50 nM [ 3 ~ ) ~ ~ , in the absence or presence of competing ligands at the concentrations shown.
. I,, represents the concentration of competitor which di,splaces 50% of thehexchangeable, specific binding of -
- - L 3 ~ 1 G A o from fie-bin~g+rotein. - - - - - - - - - - - - - - - -
COMPETITOR (pM)
. . I
Figure D3. Exchangeability of [ ) H ] G L , b'i.nding to G-25 eiuate. ,G-25 eluate was incubated with 60 nM [ 3 ~ ] G A , . After Z L h a portion of the incubation mixture was added to a test tube , containing 5 MM GAe. Samples of' both inchbation mixtures were assayed at bhk times indicated (A). he data were used - to determine the half life-of dissociation (B); /-
Time (min) r
.-concentration of a competitor that produces 50% displacement of --
exchangeably bounz [ 3 ~ ] ~ ~ , . ~ h e I ,, for GA, was about 50 nM , -
- - rwhi~eas ihat for G A , in separat& experiments was between 2.5 to . -
6 . I .>
5.0 c t ~ - (Fig. D2a3. SOP-"ligands, such as~GA&,,'were unable to
i reach I ,., at concentrations as high as 0.25 mM ( ~ i g . ~ 2 b ) .
v + By determi-ni'ng the I,, value for a series of GAS,. GA
I
derivatives and other'hormones, an affinity ranking of-ligands =
-" i
< - - - -
'for the [ 3 ~ ] ~ ~ , binding protein was established (Table ~1j.
~ f f i ' n i t ~ was hiyhest for 7-&actonic C-19 GAS with a 3 P-OH with
or without two methyl groups at C-2 (GA,, DiMeGA,, GA,). There
were nu noticeable differences between the I values for these
ligands. ~dditional hydroxylation of C-16 in the D-ring (GA,),
of C-13 (GA,, GA,), and of C-12 (GA3,).in the C-ring ~
progressively impeded binding. changes in the hydroxylation
pattern of the A-ring either curtailed binding affinity or
completely eliminated it. Thus GAS with"a 2,3 epoxide (GA,), a 1
3
exchangeable [ 3 ~ ] ~ ~ , binding. Lack of a 3 0-OH (e.g., GAS and B
GA,) resulted in greatly decreased affinity. If this lack was - - coupled to the addition of a 2 a-OH (GA,,), or the hydroxylation - of C - 1 3 (GA~,) and at C-18 (GA,,), binding affinity was lost. a . -
8 ,
The structural-specificity of the binding protein is furkher
illustrated by 3-ppi-GA, which with a 3 >-OH shoied a -"
10,000-.fold lower aff inity tha; GA, with a 3 6-OH. The sright - - - - - - Lpp - - - - - -
[ 3~]GA, displacement' observed could be a resilt of the pre,sen'ce .J
of 0.7% GA,, as a contaminant in the 3-e.pi2~A.-, as determined by 7 r . <
1
able Dl. Relative in vitro affinities of various liqands to - -%]GA, bindingprotein and their relative in ,viva
biological activity in the cucumber hypocot3 bioassay. Concentration of [3HIGA,, in the incubation m i x t u r ~ was 50 nta and that of competing ligandJ ranged from 50 nM to 0.5.m~.
1 except for GAS with superscriptc. Relative affinity was measured using I,, values determined for each ligand using the DEAE filter paper assay.
' a >. 4 u
.. I ,, competitor compet i tpr -
-2
50 nM GAu (+++) .a DiMeGA, (+++I ,6 GA, ( + + + + ) r -
0.5 pM GA, ( + + ) - - -
5.0 pM GAI ( + + ) , GA3 (++I ,
50 pM GAS ( + ) , GA, A+++). GA,OC ( + I ' - 0.5 rnM ' 3-epi-GA, ( 0 ) , .
>0.5 mM GA,ME LO), GAuME (0)) GA,' (+I, GA,ME ( O ) ,
9 GAB (O), G k 1 3 (01, G A l h (O), GA,, ( + + ) , ~ 4 1 6 ~ - ( + ) , GA20 ( O f ' ; GA22 to), G A Z 7 ( O ) , - c
~ ~ 3 6 ' ( + + + ) , GA~O' ( + + I , ABA (01, IAA (O), kinetin (0)
a~elative..activity in the cucumber hypocotyl ~ i d ~ s s a y ( + + + + , = * very high; +++, high; ++,*moder_ate; +, 10.w; 0 , vedy low or inactive) aiter srozier and Durley ( 1 9 8 3 ) ~ e x c e p t for those with "P
upebscript or . 62elative -- in vivo activity a h 4 H o a d -- et a l . 1 tombt tit or assayed in vitro at concentrations ranglng from 50 nM fo 0.25 . . -
GLC -
. . GA,,, GA,,) showed no binding affinity. other hormones such as- '
t ABA, IAA and kinetin also showed no binding affinity.
In reciprocal experiments ;sing [ 3 ~ ] ~ ~ 1 and unlabelled GA, c.
* ,
or' GA, as competing ligands, fraction containi~g the binding' 7 - . +
protein again showed a greater affinity for GA, than for G A , . , . I
GA,ME did not- dis~lbce the exchangeable [ 'H]GA , (Table D2):
- Table U2. Effect of unlabeled competitors on displacement of
I3H]GA,, and L 3 ~ ] G A l from GABP. D a t a represent dpm b~un'd-ml-~l = .
7,100 33,400 - 58 nM [ 3 ~ ] ~ ~ , + 5 PM GA,, 4,000 18,400 - 4 5 + 5 PM GA, - 2 4 ,>600 2 6 + 50 pM GA3ME - 29,100 1 3 . - - - -
40 nM [ 3 ~ ] ~ ~ , + 5 uM GA, + 5 p M G A l + 50 PM GA3ME
a ~ - 2 5 e l u a t e was assayed a t 4.2 mg 'protein=ml-l
I
I 1 1 . ~ m n
- - - -- - - --
The GA structure--binding affinity data point to the
presence of strong hydrophobic environments in the active site *
of the receptor proteih corresponding to the 1 a, 2 a and 0, 3 I i
. a , positions of the C-19 7-lactonic GAS. They also suggest c
strong'polar, or, ionic interactions in the vicinity of the ii
3 0-OH, the y-lactone ring, and the C-6 carboxyl, Weak - - -
- hydrophobic regions are llkely present near C-12, C-13 and C-16.
My conclusions about the active site of the GA receptor in
cucumber agree w-ith those obtained hy Serebryakov -- et al. ( 1 9 8 4 ) , d -
-who based thei'rs on results obtained from -- in vivo assays using
selectively modified GAS (see Fig. B l ) . However, my ,results 3
differ from theirs in the following aspects. While their data
suggest obligatory hydrophobic interactions in the vicinity of I
C-11, C-12 and'c-13, my results indicate that polar groups at - -- - i-- -- - -- -- - - -- - - - - - - - - - -- -- ---
these positions on the ligand reduce affinity but do not prevent
binding. Furthermore, I propose the presence of a nonpolar
region corresponding to the a plane of C-1, C-2, and C-3 which
those authors did not examine.
My - in vitro data on the relative affinities of GAS and GA
derivatives for the [ 3 H ] G ~ , binding protein generally support
the activity ;ankings of thk ligands in the cucum'ber hypocotyl
bioassay. At the same time, they point to some refinements and
important exceptions.
have lower activities than
(Crozier and Durley 1983) . . -
C3~]GiI4 binding protein has an approximately 50 to 100 times &
lower affinity for GA, and GA-, than for GA,. GA,, with a 2 0,-OH,
with f - 3 and C - 1 3 are re - GA, or GA, with only a C-3 OH
- - - - - - -
My binding data indicate that the
and C-7 methyl esters of GA3, GA, and GA, are reported to have
no activity -- in vivo. My binding data likewise indicate that 2 a .
- 0-hydroxylation as well as methylation of the C-6 carboxyl lead -- - >?
to a_"complete loss of binding affinity. GA, is reported to have
slighily higher activity than G A * and D ~ M ~ G A . in the cucumber
bioassay (Hoad et al. 1981), yet my vitro daka show the same -- D
-
I,, value for these GAS.
The important exceptions are GA, and GA,,. GA,, C-19
7-lacto~ic but unhydroxylated GA, and GA3,, a C-20
3 0-hydroxylated GA, are both reported to have a's high -- in vivo
activity in the cucumber bioassay as GA, (Crozier and Durley - - -- 7--- - --- - - - - -- --
1 9 8 3 ) . They are believed t&ct as immediate biosynthetic. -', c
precursors of GA, in cell free'extracts and shoots of cucurbits
(Hedden 1983, Graebe -- et al. 1980, Yamane 1987). My data show
'that GA, has very, low and GA3, no affinity for the ['H]GA,
binding protein - in vitro. Possibly GA, and GA,, have high
activities -- in vivo because they are metabolized to the active GA
in cucumber, pr~sumably. Ga,. +
GA,,, a C-20 b-lactonic GA, is also reported to have - - - - - -
moderate activity in the cucumber hypocotyT--bToassay (Crozier
and DurJey 1983 ) . My data indicate, however, thaY the [ 3 ~ ] ~ ~ ,
3
binding protein in cucumber has no affinity for G A , , . The
moderate in vivo activity of Gk,, may be due to the possibility -- that in soIlction it occurs in equilibrTUKwith its .C-20 alcohol
"
open lactone form which is reported to be a precursor of GA.
(Hedden 1983). v
The observed structural specificity of the [ 3 ~ ] ~ ~ , binding
protein is common for proteins but the fact that some of the
precursors of GAu biosynthesis in cucumber, namely GAG and GAS ,----
have little or .no affinity for the binding protein suggests that
.we are dealing with a receptor protein rather than an enzyme of
GA metabolism. An inv~lvement og emymes, suck as gfjycosylztses - - -
(~chneider 1 9 8 3 ) in thea obssrved binding of [ 3 ~ ] ~ ~ a has,
however, not been excluded.
P The - in vitro assay of thedisplacement of radioactive GA
binding to cucumber cytosol under conditions o'f minimal GA
met a h l i s_m_ i s a pswyf ulLtec hnique -to- determine those-pacts -of----L
I -the GA molecule which are 2mportant for a structural fit to a /
putative receptor. However, the technique is l?mited by then 1 I , !
purit,y of 'the radiolabeled GAS and competing ligands and an I
accurate determination of their mass. Also, to exploit the
t-echnique to its fullest potential, the proportion of specific
to nonspecific binding, which in turn is determined by the
purity of the binding protein, should be,as high as possible. In
- - -- - - - - - -
total binding and, as mentioned earlier, the [ 3 ~ ] ~ ~ u . was about
61% pure. Efforts were therefore made to increase the purity of
01 t n e s l n l n g protein.
PART E
Partial Purification of a GABP Fraction.
1
b. --- I
The results from Parts C and D support the view that the
this hypothesis and identify the cellular functibn of the GABP, ,
d
pure preparations of this pr'otein are required. Efforts were
therefore made to purify the GABP from'cucumber extracts. For
thjs purpose, salt fractionation, gel permeation, ion exchange
, and hydroxylapatite chromatography were examined. A number of
other separation techgiques were alsos tested. 0
%
I . Materials and M e t h o d s . 1
C ? * .*;. r I
- -
1 . Chemicals and materials .
I
The source and purity of the GAS used has already been 1
described in ,Part, C, ater rials and .Methods. ,
~ e ~ h a d e x G-25 SF,,. G-75 SF, DEAE sephadex A-50, and Sephacryl - k - . --
S 200 were purchased from Pharmacia, ~ ~ 3 2 ' and- DE51 anion
exchange celluloses were products of Whatman. Hydroxylapatite -
high resolution was bought from Calbiochem, and blue -
dextran-agargse (Matrix Gel Blue A),from mic con. ~ l l
chromatography meterials were equilibrated and packed into
' columns as prescribed-by the manufacturers. All other chemicals >
were of analytical grade.
n
The following buffers were used:. '1
b , b.
A , 100 I?&! Tris, 1 mM EDTA, 5 mM dithiothreitol (DTT), 50 *
PMSF, adjusted to pH 7.3 with H,FQ,; - 9 . (
B, 20 mM Tris, 1 mM EDTA, 5 mM DTT, adjusted 'to pH 7.0 with
. . D, 200 mM K-phosph&e, 1 mM EDTA, 5 mM DTT, pH 7.0;-
E, 25 RIM bis-Tris, adjusted to m6.3 wit+ Hei; -
F , 10% ( v / v ) Polybuffer 74 (~harmacia), adjusted to pH 4.0 with -
20 mM bis-Tris, 5 mM DTT, adjusted t o - p ~ 6.0 with HC1; , . I - - --
- t
2 0 mM piperazin-mM . DTT, . adjpsted to pH 5.5 with HC1; *
buffer H ,adjusted to pH 5.0. 1 t
i - , , \
<.
GABP - extraction - and purification a
C a d +
Hypocotyls were harvested into bulffer A from seediinijs grown - . f *
h - described in Part C. For-large scale extractions excseding ' - u
. <
,200 g of 'hypocotyl tissue, sect.ion.s were homogeni'zed with 5 I . - .
bursts of 4 sec each in a waring7-blen.der in *an equal volhme p
f
( I : \ , w/v) of buffer A, otherwise a mortar and-bestle were used: '
The homogenate was filtered through 2 -layers of nylon screeniag
and'the filtrate centrifuged at 100,OOOqf+ 1.5 h, 'to yield the
cytosol. (NH,) ,SO, was added t o the cytosol as described in
'~esults' . After equilibration and centrifugation at' 23,00Oq,- 20
particulates removed at 7 , 0 0 0 ~ ~ ~ 5 min. The supernatant was , I-.
desalted on"a.Sephadex G-25 SF column (2.5 x 16 crn). In early
'experiments the G-25 el-uate ;as stored as a lyophilized powder
at -30 C. Later it was stored at -70 C after ireezing in liquid . , . N2 i
6 ?
Pooled 6-25 fractions were loaded onto a DEAE-ion exchange ~
colLmn (DE32. whatman) aqu'ilibrated in buffer B and eluted kith $5.
a gradient of K C 1 as Mer'i-bed i-n -'-Resul%sL.- The G ~ B P + w ~ k ; t i - ~ i f t c ~ - - - I
f raction,was precipitated qwith 70% TNH, ) 2S0, and resuspended in
i
*- -
- G. I
u
buffer B . -- -
- - - - - - - - -- - - - -- - - - - - ' A number of app~oaches were'tesfed fox increasing the purity -
4 - - \
of this DE32 eluate: - . , k a
\
a. Hydroxylapat i te chromatography. G - ~h'e matrix consipts of calcium phosphate crystals and acts
4
s'imilar t b an ion exchanggr but 'also tends to bind cglcium I
9 - 4 pbinding4*iproteins; DE32 eluate 'in buf fe; C was- appl ied to- ; 2.5
a -
- 8
crn x, 15 cm co'lumn: and 'unbound .protein washed out. GABP was % .' v
eluted with a single step gradient of buffer D. The eluate w~ - --
- --
precipitated with 70% f , N H , j 2 S 0 , and resuspended in a small. . . a
volume of buffer B. It cduld be stored a.t -70 C after irkezing + L 0
- - in liquid N,. -
L
+
b. Dye-ligand chromatography
- <
One-half ml of DE-32 eluate (containing 2.4'mg proteinr in :,
buffer B was allowed to adsorb for\ 30 m<n to a blue *
dextran-agarose column ( 1 cm x 3 cm) . The matrix 'used has a - ..
tendency to bind dehydrbgenases and ki~ases, as well as ~Sorne
other proteins ( ~ n o n . 1979),>. Unbound protein was washed o u t with
10 ml buffer B. The bound' GABP was eluted with 10 rnl 1.5 M KC1
in buffer B. The eluates from several such separations were
combined, precipitated with (NH,),SO, and resuspended in b u f f e r
B for assay.
a 7-
. . V) d , c-
. _ , G e t permeat io; rhmimrography was performed uslng - L. a waters : 'f -
chromatograph ri t h 2 M5lO 'pumps, a ~ 6 ~ ( 3 a d i e n t c o n t r o l l e r , and
, '- a M740 r ecorde r / in fegra to r a t room temp"eLrature. I 125 a&--300 SW * b .
( w a t e r s ) gel f i l t - ra t ion columns codet ted i n seri-es were. used: I
Pi
Samples ('100 u l ) were e l u t e d . w i t h buf fe r D a t 0 . 5 m l - r n i n - " . , ~ l u e ', I - .
,dextran, f e r r i t i n , c a t a l a i k , a l coho l dehydrogenase, BSA, -kb - - - -. - --
o u a l d u m i n ~ a n b cytochrome'c were used f o r molecular weight C
c a l i b r a t i o n .
- . d. Fas t pro te in l i q u i d chrmatogrdphy -
For chromato•’,ocusing and _ a n i g n &charige chromatography a - . Pharmacia FPLC chromatograph-with 2 P500 pumps and a LCC500
1. I -
grad ien t c ~ n ~ r o l l e r were used a t 3 C .
\
Chromatofocusing. A Mono P HR 5/20 column (Pharmacia) waS 4
- - - - - -- - - - -- - - - - - -- - - - -- - - -
e q u i l i b r a t e d - with b u f f e r E . Immediately p r i o r t o sample lohding
a 3 m l a l i q u o t of buffer F was passed through t h e column. &32 1
< -
e l u a t e was e q u i l i b r a t e d with buffer'^ using a small d e s a l t i n g 2, *
column. Four m l of kluaTe m n t a i i i i 5 - g 6 t o 8 mg p r o t e i n were 1
a p p l i e d t o the column a t 0 . 3 ml-min-.'. Af te r e l u t i o n of unbound
p r o t e i n s , \ .
ml-min ' . Fract'ions
r Anion
buffer F was passed through t h e column a t 0.5' . .
This res;lted in a PH c~r6d ien t from pH 7 t o 4 . .
were pooled fo r a s say .
- -- , - exchanqe chromatography. ~ a n @ l e s - w e c chromatographed
a prepacked Mono Q HP 5/5 (quaternary amino, e thy l ! ion I
4 . - - - -- I n
, . . , 1 , . - I p. mi . . 0 -
4 -, . exchange column' (~harmacia)..' Chromatography w a - s performed at pH - 4-
L- b - -
7.0 w&h buffer 0 at pH 6 -
, o c
--> - - -I I.
. iith b;ffers H da'nd"1, F
w'ith up to 1 M KC1 .in these buffers. Y
F
\ w
i b 4. - In vitro GABP assays \
- -- - A S was shown iiLparf C, many rna~~ornobecu~esi bihd GAS-
resulting in apparent specific binding. In order to discrimi~ate - 6
. between this .and receptor-Cype specific binding, each fraction* . d
was assayed with f 'H&A, in the absence and'presen-ce of a 100 -
fold excess of ,unlabeled-~~, (,high -- in vivo activity),
(moderate -- in vivo activity), and a 1000 foid excess of-GA,ME (no 0
in vivo .activity). The resulting displace men^ of [ 3 ~ ] ~ ~ , from 7-
B
th'e binding sites by the competitors was used'as a guide to
select those fractions containing the putative receptor. In'
2 -t-o correlate with -- in vivo activity of the competitor. Eor most w . ,*?
experimenfs 50 nM [ ~ H J G A , prepared from NEN reattion products
was used. However in later specified experiments, 1 0 ' h ~ [ 3 ~ ] ~ k , - . purchas'ed from Amershdm was used. At this lower' concentration, ' _ - bacl!ground binding yas reduced and the GABP could be detected at
\ 'sample protein concentrations as low as 0.2 mg protein:ml ' , &,5~ y- a - I thereby allowing incre sed assay sensitivity. y:
~riplicate 100 p1 samples o•’ incubation mixtures were
assayed with the D E A E cellulose fiiter assay as described in
I
' Samples were desalted using a small Sephadex G-25 column.
, The protein3iractioh >as precipitated at -20 C for 1 h with 9
volumes of 1 0 mM 0-mercaptoethanol i,n acetone, Precipitat-es were
pelfgted at 28,0ffOg,-5 min, washed and then -xashe&twi-ce- w i t h 4
anhydrous ether. The pellets were resuspeqded in ~aemrnli's s
, sample loading buffer (~aemmli 1970) and boiled 'for 5 min. - iUectrophor&is was performed with 12 mA constang C U ~ M R ~ at 2 4 -=- - -
t
using a commercial slab gel apparatus (model SE 600, Hoefer ".
Scientific Instruments, San Francisco CA). One and a half mm
thick gels (?separating gel: 8.5%T, 2.7%C; stackin-g gel: 4%T, &
2.7%C) were
et al. ( 1 9 8 --
used. Gels were silver stained bed by Wray
- i;,II.lts >,,p - . -> ib
. J b I -
-- -- -
- I . Source - of tissue extract ,
. - specific binding was dbserved in G-25 eluate prepared from .
dry Cucumber seeds as v e i l as seeds germinated in runiing water b - .
for up to 96 4 (Table El); However, on a mg protein and a fresh
weight basis, hypQcotyls_ of 7 d a y old seedlings showed greater- ----- * *
?amounts of ectractable sbecific binding (Figs. El agd E2). t .
,
Furthermore, target (apical) as well as nontarget (basal)
., regions of the hyp~cotyls of 7 day old seedlings bound G A L + - -*-
specifically i~ig. E2). On a per mg protein basis both the $
- apical and basal regions had a similar number of binding sites
(n = 0 . 1 pmol-mg-' protein) and a•’ f inity (Kd = 30 nM) f b r GA. . I
However on a fresh weight ,basis, 6 times more GA binding si" f es were observed in the apical tissues (n = 0.2 pmol-g-' fresh
-- L-
we l gm t compa redtrthinintargetEgi on T n P = T .X3- pmol-g-' fresh weight).
Although harvesting api<zl hypocotyl sections from 7 day bld
seedlings is very labor in~ensive, this material was chosen for . __- -.
GABP purification. It was expecked that purification of this. /
protein would be facilitated By'usinq source tissue which is
enriched in this protein, or by excluding tissues which have a
high concentration of other proteins. - - -
I ~
a
h
l
D
\
I
t 5
(
+I
'
, -
I -
* t I
Table El.
[ 'HIGA, bi
nd i
n^ to G-25 e~luate froA, whole se~ds germiqated for
e
of time and from apikal parts o(f hypocotyls from 7 day old seedl'ings.
I
Seqds were germinated in runninlg water,for theduration shown.
2,
extracted in
the same manner
ad
7 day old hypocotyl sections;.
[ '@
1GA
l (Amersham) bound*mgl protqinG; All samples were
' . concentration of 2 mg*mL-'.
I I
\
#
'I
I .+y
I I
I .
,
d
1n~ubat
i'on
, seed germination ti
me
d
hypocotyls
~*
Oh
8h
16h
24h
48h
96h
7 days
I 1 '\
~ 19
nM
[ 3H!GA,
0.17
0.18
'0.15
0.21
0.15
0.17
0.29
*
v
L a,
'
'J,
a;
+
1 P
M GA,
0.06
0.07 0.OF
0.11
0.06
0.09
'0.09
- u
I I
03
I 0
*
- .I
I a.,
I I
,
I +-
I I
1
I I
- .
I I
I I
,
I
I I
I
4
I I I '
I I i
I B
-+ a,
I
I..
. #
.. I
..
I 4
-
I 0
1
-
1 e
I'd
I 1
B
I I
I
"
, I I I
b
\ I
n
b ~
i *.
I I t
I
a9
-
I , .
I I1
I
~ i g u r e El. Scatchard plot of specific binding of GA,, to G- d
rom ungerminated seeds.
Amersham [3H]GA, was-used. The data point at ' * ' sugges
the presence of a second class of binding sites-with a
Figure E2. Scatchard plot of specific binding of GA," to cytosol i from apical and basal hypocotyls of 7 day old seedlings. \
\
Z2JCEhct of dithiothreitol . . F ' -
4 ?. '.
e
Dithiothreipol (DTT) is a reagent for the protectbn of % * 'I
sulfhydryl grou#s of proteins (Cleland 1 9 6 4 ) . Its effects on . *
levelsand stability. of receptor-type binding were tested. * results - - shown in Tables E2 and E3 indicate-that the
E: -/ -- , DTT throughout the GABP extraction significantly enhances
- specific .binding- befor& and af ter--Sto~age at--10-X-.-Theef &ct--------
IS i
ersib"1e as its removal from the extract results in a loss 7
of 'this protection. Addition of DTT to a GABP extract,'which had
been p&a~iy purified in its abb_sence, rssul ted in a a l i g h t -.: F
enhancement of receptor-type bi-nding. Five rnM D?T offered the '
same protection a's 10 mM DTT. All. purification buffers therefore
includ%d 5 mM DTT.
' 3. Anynonkum sulfate precipitation 1 - - - - - - - - - - - - - - - - - - - - - -- -
0
Differences in the charge characteristics of proteins
affecting their solubility in (NH,),SO, solutions -were"exploited - -
for the cbncentration and partial-.purif icati~n of the GABP from . -
cytosol,. The experiments 'in tho previous chapters were performed
using G - 2 5 , eluat;;' prepared from cytosol precipitated with 60% *
fNH,),SO,. I t was attempted to reiine this step further with the '
next experiment. A i
- -
L --
The I ~ U , ~ O U ~ ~ C ~ S O S O ~ ~ ~ 3ipocotyl extracts was divided into
5 portions. (NH,),SO, was added to each portion to yield J
- . - - - - - - - - 4
-- @% - - - -
I
-- .*- a i
.-, ~
B 3 .Bjp! t of pr,.lfenc, -- , of DTT ndlng to $
eluate:, . . <, G-25 e lua te from hypocotyls Jas d.>eing e x t a c t i o n
~ ~ = ~ f - e r - ~ ~ ~ c K l - n g - ~ n - o D T T ) or con t a i n i II T u mM d i t h i o t h r e i t o l (DTT throughout). In one r x t r a c t ZTT was removed from the (NH,) ,SO, pe l l e t i n desa?ting s t ep (DTT removed). Data -sepresent.r3H]GA, pro te in) .
f -
* -Z - -
Incubat ion' noDTT DT b4-hpughout DTT removgd - .- 9
50 nM [ 3 H ] G ~ , a 0.10 0.16 0 - 0 9 + -5 pM GA-,-:- - - - 0 a - - 0 05 - 0- 0 5 *. + 5 pM GA, - 0 - 0 7 a$ " 0.07 +50 pM GA,ME -0.09 - 0 . ' I 4 - " 0 .10
- - \ - - - - - - L 7
d .B
~
+ . " Table A E 3 . - E f f e c t -of 14 _rnM DTT e n s t a b i . l i t y of G-25 e.luate st
a t - 1 0 C f o r 3 days. - * A
,G-25 e l u a t e 'from e x t r a c t s prepared a s descr ibed i n Tableb=E13 \ ' was used f o r , storape : ~ T T add&.' was p~epared hy adding, nT-T - . . t o 'no DW' e x t r a c t upon t-hawing. Data- represent I 3 H ] ~ ~ , . . bound (pmol-mg" p t o t e i n ) : - .
8 - 5, * @ Incubat ion , ~ * - D T T DTT throughout DTT added $
rr
C
50 nM [ 3 ~ ] G ~ , ' O., 1 1. 0.13 4.. 08 + 5 PM GA, 0.07 . 0.04 0.05 . + -5 PM GA3 - 0.07 0.09
-- --
0.07 ,
+ 5 0 jiM-GA,ME - - - - -- - 0 % 1 0 " O T ~ - -
- 1 -- * -
.. concentrations of 30, 40, 50, 60, and 85% saturation. The
I - /
pellets obtained after centrifuga.tion were desalted4 by gel A -
, E4) indicate that the GABP precipitates out at an (NH,),SQ,
concentration of 40% or more.,$n another experiment, (NH,),SO, - d b s added to cytosol to sequentially reach 30,- 40, 50, and 60% .
- sa-turation. pellets obtained at each s t e p were removed for r i ' J
." assay. As shown in Table E5, the - putative receptor appears - - - to - . -- - - - s
precipiaate lout with 30 to 60% (NHU );SO,,.Qith the majorit* I
- preci-pitating with 50% (NHq)2SOi.vN~ GABP was detected in:60-80% *
(NH,) ,SOq pellets (data not shown) .- Only 1 to 10% of the tdtal.
cytosol proteins precipitate out with the 30% (NH,),SO.. For - 1
A E
this reason, .and to save time, cytosol was precipitated with 50% - -
(NHq),SOo in early experiments. In later-experiments, however,
cytosol was precipitated with 55% (NH,),SOI after the 30% pellet
was removed.,This allowed increased' yield and rqa~val of some
- - - - - - - - - - lipids and hydro~hobic proteins which tend-to adsorb:, to ,
chromatography matrices in-,subsequent purification steps. 1 1
Precipitating GABP over a narrower range of (NH~)~SO,,
concentations does not appear to be practical.
About 50% of the total cytosol proteins are
preparation with this purification step. -
removed from the f - \
v - 3
1. "
m b
(NH4 ) . 2 S 0 4 . t
--
Cytosol was divided into 5 aliquot~. (NH,),S0, was added to A-
each f r a c c o n to-givethesaturat ion shown: Each pel-let obtained was desalted psior to assay. Data represent [ 3 ~ ] ~ ~ , '
bound (pmol.mg:' p r o t e i n ) . - , . B -
Incubat ion Buffer B (NH,),SO, Pellet . 30% 40% 50% 60% 8 5 %
' 5 0 nM [ 3 ~ ] ~ ~ , 0.31 0.95 0.31 0 .30 0.30 - 0.34 + 5 VM GAQ 0.13 0.37 0.18 0.19 0.23 - f i . ? f L "
-
- - 0 .37 0 .23 0 .20 0.21 0 . 2 3 - + 5 UM GA3 0.13 +50 pM GAQME 0.12 0.38 0 .30 0 . 3 0 0 .29 0 . 3 2
e z .
0 * .- in - -
r GA t o cv toso l fract ions4 seauent i a l l y / -- Binding nf I 4 A .,
c u t with (NH4)2S04. Cytosol p r o t e i n s were p r e c i p i t a t e d with 30% (NH,),SO.; The ~ z M t a i n & * s F C ~ J ~ N f o r a w l ' while ' t h e supcrwt ' was f u r t h e r f rac t i 'ona tee with inc.reasing (NH.,) ,SO4 s a t u r a t i o n as shown. Each p e l l e t obtained was d e s a l t e d p r i o r v
t o - a s s a y . Dam-represen t [ 3 H ] ~ ~ 4 bound (pmol-mg-' p r o t e i n ) .
1 '
I ~ n c u b a t i o n Cytosol (NH,),SO, P e l l e t
30% 30-40% 40-50% 50-60%
. . - =.
#
. t '3
\
C
4. Open column 5on - exchanqe chromatography \
- - - - - -- - - --- QI &
A number 'of ion exchange chromatography matrices were
tested, including DEAE SephaceL, DEAE ~ephadex A-501, DE32, and * 1
DE51. The DE32 ion exchange cellulose had the best flow
.propertie,s and gave the best chromatographic separation of G - 2 5
eluate. Two column sizes were used. FO~'.G-25 eluate containing . . - - - - - -
up to 70 mg total a 2.5 x 13.5 crn column was-used: for
large-scale preparations containing up to 400 rng protein a 5.0 x I I
16.5 cm column~rovided optimal separations. Typical elution -
profiles for these columns are shown in Figure E3. When loading -
samples in buffer B , all r'eceptor - type binding activity was . ,
retained in the.column. GABP could be eluted with -0.23 to 0 . 3 G M
KC1 using a linearqradient of 0.18 to 0.33 M KC1 (Tables E6 and '
I
E7). About 18% of the total proteins loaded onto the column
in about a 4-fold enhancement of the specific binding detectable
in the G-25 eluate (Table E8). '
5. Further purification -- of DE32 eluate \
- a. Hydroxylapatite chromatography I ,
A slight enhancement of sp&ific binding was achieved by
passing the - active - fraction from the DEAE cellulose through a
form the matrix of this column. Differences in charge and
calcium binding properties of proteins are exploit* with this
, t
Figure E3. Elution profiles of and 5.0 x 16.5 cm'(b) DE32 Regions marked A to F', were
w The fractions labeled A' to _ those in
G-25 eluate bn 2.5 x 13.5 cm (a) anion exchange columns.
for GA-binding activity. ) are not equivalent to
- Elution ' Volume (ml)
B
Elution Volume (ml) rl
1 *- -
. -
Table E6. Birding of [ 3 ~ ] ~ ~ , to fractions from a 2.5 x 13.5
-- -
cm DE32 anion exchange column. Pooled, lyophilized f r a c t i o m collected according to
-- - A - E3a, were used for the -incubations. Values qiven repr [ 3 ~ ] ~ ~ , bound (prn~l*rng-~ protein). All fractions were assayed at a protein concentration of 2.2 rnggml-'.
A -.
Fraction A B C D E
50 nM [ 3 ~ ] G ~ , 0.30 0.13 0.17 b . 2 7 , 0.38 ' - 0 . 0 9 + 5 pM GA, 0.20 0 .09 0.08 0.11 0 . 1-7 + 5 p M GA3 0.20 0.09 0 . 1 0 ' * O . t 5 0.21
- J -
+5OPpM GGquME - 0 . 1 9 0.09 0.11 - 0.22 0.30 ' - 0 . 0 8 -
8 -
Table E7. Binding of [ 3HIGAa to cy-tosol, G-25 eluate, and fractions from a 5.0 x 16.5 dm DE32, and a 2.5 x- 1 4 . 8 cm
- - n, - :;% ;:::ions were collected a,s shown in' Fig. E3bi frozen in liquid N,,and stored at -70 C prior to assay. Values given represent 1 3 ~ ] G ~ , bound (prn~l*mg-~ protein). All fractions were assayed at a protein concentration of 2.9 mgaml-', except cytosol- (1.3 mg-ml-') and HyAp unbound (0.8 mg*ml- ' ) . s \
cytosoL G-25 DE32 - - HYAP
- Fraction e s u a t e A B ca un- 5 o u n a - ,.+
- bound -
50 nM [ 3 ~ ] G ~ 4 . 0.15 0.08 0.06 0.09 0 .23 0.13 0 .26 ,
+ 5 pM GA4 0.09 0 . 0 3 0.03 0.03 0.03 0 .1 1 0.04 + 5 pM GA, 0.02 4 . 0 7 0.06 0.05 0 .13 0.09 0 .14 +50 ~M'GA,ME 0.17 0.07 0.04 0.08 0.79 0.11 , r . 2 3 -
45:
was
, .
loaded onto the hydroxylapatite c o l u m n , a - . .Lr
, . 9
Table E' if icat ion of GABE -from cucumber C1
01 Spec8~f?activity values calculated from ~~~~spr;sented in Table E7. The difference between [ 3 ~ ] ~ ~ , binding in absence . .
- - - - --
7 o n s becau ca f lc bl
displaces a large amount of radiolabel in this fraction (See . Table E7). This results in a greater than 2-fold " underestimation of the extent of purification and recovery achieved.
5 -
Fraction total spec i f ic total purif i- reco- .protein activity specific cation very_- -- - - -
(pmol per activity (mg) mg prot.) ( pmol ) ( % ) k - q
cytosol ~8 7 2 0.06 ' 52.32 , 1 ' . 100 G-25 eluate 384. 0.05 19.20 1 37 DE32 72.4 . 0.20 14.48 3.3 / 28 HyAp bound 39.9 0.22 8.78 3.7- 17
-
1
method(Gorbunoff and Timashef t 1 ~ 8 4 ) . As' Shown in Table El,, the ,
-- GABP is bound by the HyAp if % the samples 'are loaded in buffer F. t. 4
The GABP can be eluted with a single step gradient using buffer --
G. This procedire results in only 1 Q % improvement & specific binding of the DE32 eluate. About 10% of the proteins loaded
onto the column are unbound and thereby are removed from the
GABP . CC
- - - - - - - - -- - - -
Table E8 shows khat with this procedure the GABP was d
purified about 3.5-fold, with about- 17% recovery of the specific 4
binding present in cy,tosol. - - --
A GA, binding to the HyAp .fraction*is saturable (Fig. E4). --
Scatchard analysis of the HyAp fraction suggests the presence of
a single class of binding sites with 0.6 pmol binding'sites.pPr
mg protein and a Kd near 50 nM ( ~ i g . E5, compare with Fig. ~ 2 ) . %
The differencrs in Kd between cytosol and the HyAp extract may
n%t be significant. They may also be related to- the use of
different sources of [ 3 ~ ] ~ ~ 4 for the different samples. *.
1
The elution profile of the HyAp fraction from analytical A
runs on combined HPLC gel permeation columns is shown in Fig.
E6b. Comparisoq of the chromatogram of the HyAp fractign with
that of the DE32 eluate i'ndicates that HyAp enhances the , C -
proportion of a high molecular weight ( > 4 0 0 kdalton) and a 110 , -
kdalton protein (Fig. E6). Figure E7 shows the SDS-PAGE profile L 1 - -- - - - -
of this extract. Compared to G-25 eluate; the-&^^ fraction -- '
again shows an enhanced band at 110 Kdalton. However, peptides 4 --
-
Figure E4. Saturability of GA, binding by fraction. s
The HyAp-bound fraction was incubated at the concentrations shown-(Free). A
the HyAp-bound
with Amersham [ 3 ~ ] ~ ~ , lV00-fold excess of GA,
was used for deriving specific from nonspecific and total binding values. The extract was assayed at a protein concentration of 0.7 mg=ml*'.
~ ' l ~ u ~ e E5. scatchard ana ly s i s of HyAp-vnd Amersham [ 3 ~ ] ~ ~ , was used. . I
L I
I
f r a c t i o n . -
Figure E6. Elution profile of DE3 d eluate ( a ) and HyAp-bound (b) on HPLC gel filtration columns.
- -
- - -- -- - --- - - - - - - - - - -- - -- -- - - - --
No. R,(ml) Kdalton 1 , 1 1 . 1 389
Figu re E 7 . SDS PAGE of G - 2 5 e l u a t e and HyAp-bound f r a c t i o n . Molecular weights of some o f t h e bands d i f f e r i n g between t h e s e e x t r a c t s a r e shown. >
-- with a moleculareig*t7near 62, 76, a m € F K m t o n are also , ; - - enhanced. Numerous - A - - - other - protein bands are itill . apparent in the
L
HyAp frac'tion., L
Problems with recovery and column performance have
successful - further purification of the HyAp fraction.
b. Dye-l3gand chromatography - i { - -
An almost 3-fold purification of the DE32 eluate was
achieved by chromatography on a Blue dextran-agarose column. As
shown in Table E9, the GABP is bound by this matrix and ca; be -
eluted with 1.5 M KC1. However, only 24% of the specific binding
and 23% of the total protein loaded was recove.red from this
column. i
c. FPLC - Chromatofocusing
A Mono P chr omatdocus ing m M n was used_on_ theEPLC- to -- i d ,rC
- fractionate the DE32 eluate with a pH 6.5 to 4.0 gradient. On 7
such a column proteins elute at a pH lower than their PI. This
is due to their low solubility at their pI and Donnan potential
effects near the chromatofocusing matrix -- buff%er interface
- (Anon. 1982). A typical eluti6n profile (Fig. E8) shows that a l i / = 6
large proportion of the proteins-in this ext-ract elute bktween --
pH 5 and 4. The - in vitro assay of e-luate fractions (~able ~ 1 0 )
indicates that the GABP alsp elutes with the majority of the - - - - - - - - - -
, proteins. Only a slight enhancement of specific binding was -
observed in this fraction compared to the starting material.
Table E9. [3~]GA, binding to DE32 eluate and fractions unbound ,
. an& hound by--Blue dextran-agarose. 8 Fractions were assayed with Amersham [ 3 ~ ] G ~ , . . - Values
r e p r p s p n ~ J 3 ~ 1 G A , hamd I g m l a q - ' protein). - a
- -
- DE 32 Blue A-agarose Incubation b unbound bound
Figuke E8. Elution profile of DE32 eluate chromatofocusing column. Fractions were callected for assay as
from a
indicated.
- Elution volume (ml)
li
:'
Ta
ble
E
l*.
[ 'H
IGA
. bi
nd
in
6 DE32
elu
ate
an
d
fra
cti
on
s e
lute
d f
rom
c
hro
rna
tofo
cu
sin
~
colu
mn
(
~o
no
P).
1
~r
ac
tio
ns
wer
e c
oll
ec
ted
acc
ord
-in
g t
o F
ig.
E8
and
a
ssa
ye
d
wit
h
Am
ersh
am
rep
rese
nt
[3
~]
G~
,
boun
d (p
rno
l.m
g-'
p
rote
in).
f3HlG~,; Dat
'Sam
ple
DE32
/x
Mon
de.P
elu
ate
v
A B
'
C
D
i
10nM
[,H
]GA
, 0.15
0.11
0.11
' 0!07
+
1 pM
GA
, 0.06
0.02
1 0.08
0.05
% 0.04
'+
1
pM GA,
-
0.07
0.02
0 (0.5
0.08
0; 05
+10 pM
GA
,ME
0.09
0.02
I$, 0.06
0.10
0.04
'i
- - - A- - - - - - - -- - -- -
= L
P
Only 20 t o 35% of t h e t o t a l protecn loaded onto t h i s column-
--- - - b r - 0 ~ dsaay; %Phis rnetktj, t h e r e f o r e , d i d not
appear t o be p r a c t i c a l f o r t h e p repara t ive p u r i f i c a t i o n of the
L p *
-
*a,
d. FPLC - Ion exchange chromatography -
The r e s u l t s from column implied t h a t ' - - - - - - - - -
i
' t h e GABP w i l l bind t o an anion exchange matrix even if the pH of i
t h e column b u f f e r is lowered from pH 7 t o p ~ 6 br 5 .5 . By
lowering the'pH, those pro t ,e ins which have a pI near t h e pH of - ,f --
t h e column b u f f e r would show l i t t l e binding t o the ma t r ix . '~uch ,
p r o t e i n s could thus be, e a s i l y excluded from the mixture t o be - *
p u r i f i e d . '
The e l u t i o n p r o f i l e s of DE32 e l u a t e on a Mono Q - FPLC ion -
exchange colum-H 7 and 5.9 a r e shown i n ~ i ~ u r e ' ~ 9 . %he * .
-- - - - - - - -- - - -- -- - - - L--
assay of t h e e l u t e d f r a c t i o n s (Tables Ell and E12) confirms t h a t
no GABP e l u t e s i n t h e unbound f r a c t i o n s a t e i t h e r pH. However, ' . s p e c i f i c binding was observed over a broad range of the bound
f r a c t i o n s . GABP e l u t e d w i t h 0 . 1 8 t o 0.43 M K C 1 a t pH 7 . 0 , and 0 - +
t o 0 . 3 7 M K C 1 a t pH 5.9. Bound f r a c t i o n s d id not show enhanced
s p e c i • ’ i c binding compared t o t h e DE32 e l u a t e . s i m i l a r r e s u l t s L .
were obtained a t pH 5.5 and 5 . 0 , even when s t e p g r a d i e n t s - e r e
%-. used t o ensure complete sepa ra t ion of p r o t e i n f r a c t i o n s ( d a t a
not shown) .
Pro te in recovery from t h e Mono Q column was genera l ly neark
50%. Some of t h e lokses mai have been due t o p ro te in - -
Figure E9. column
Chromatography of at pH 7.0- ( a ) and
DE32 eluate on !
Mono Q
-
Lo:
I
Elution volume
Table Ell.
[IH)GA.
binding to DE32
and fracti~ns eluted from io
n ex
cha'
nge col
(Mod0 Q) a
t pH
7.0
. ~radtions were collected according to F
ig. E9.a and assayed with Amersham [IHIGA
Datq represent. [
3~]G~,
bound (pmol-mg-' protein).
I
Incqbation
DE32
,~
Mono Q
el$ting KC1 concentration' (M)
I
0.1
8-0
.30
0
.30
-b.4
3
O,.
43-0
.67
B C
?
+
1 pM
GA
3 +10 MM G
A,ME
I
#
I I I 1
$
1"
~
Table ̂
ElZ
. [
'HIGA,
binding to D
E32
eluate a
nd fractions eluted •’rom ion
,(yono Q
) at pH 5.9.
Frlact ions werem collected accorjing to F
ig.
E9.b apd assayed with Amersham
[ 3
~:
DaltaGrepresent [
3~]G~,
bound (pmolemg-' protein).
I I I
*
Mono Q
elutin4 K
C1
concentration (M)
0-0
.20
0
.20
-0.3
0
0.3
0-0
.37
0
.37
-0.4
0
B C
D
~ E
precipitation on the column. This precipitation which was more .r
apparent at acid pH values was further mvestigated. I
- - - - - - - - - - - -- - - - - - - -
e. pH fractionation -
The precipi-tation of some proteins in the DE32 eluate at
acid pH., suggested a possibility for differentially
precipitating proteiqs at different pH values. For this purpose,
DE32 eluate in buffer B was equilibrated with an acidic buffer - i
using a small Sephadex G - 2 5 column. The pH of the eluate was -
readjusted to the desired acidity, and precipitates formed were %
removed by centrifugation. The pH of the supernatant was lowered
again and the process repeated. Proteins in the final
supernatant were, collected by (NH,) ,SO, precipitation. All
pellets were assayed at pH 7.0. - $
/
The results of the vitro assay, performed by Ms. Joan + *
Chisholm, indicated that the GABP precipitates between pH 7.b
- and 5.7 (Table E13). This was also confirmed with DE32 eluate
prepared from seeds germinated for 24 h able ~14). About 40 to
60% of the total protein mass remains in the pH 5.5 supernatant.
Lack of specific binding in pellets obtained at pH values less - than pH 5.5 does not appear to be related to denaturation, as -
. .'+ active GABP was recovered from the chromatofocusing column
between pH 5 and 4.
i ,\ ,
1
Table E13. Effect of pH on precipitation of GABP in.DE32 eluate from hypocotyls.
DE32 eluate was equilibrated into
20
mM piperazine-HC1,
5 mM DTT, pH
6.0
usipg a
, desalting column. The eluate was readjusted to pH 6.2. Precipitates forme& were
collected by centrifugation. The pH ofa'the supernatant was lowpred to the value slfiown
and ~ecentrifuged. This process was repeated several times. Proteins in the final
supernatant (F.S.) were collected by ,(NH,),SO~ precipitation. All pellets were
resuqpended in buffer B at pH 7.0
prior to assay. Data shown represent Amersham
[3~]G~q
bound (pmol-mg-' protein).
I
Incubat ion
DE32
I PH
F. S.
\
a assdyed at a
protein concentration
<0
.2 mg-ml-'
i i
Ta
ble
E
14
. P
iec
ipit
ati
on
of
G
AB
P
in
DE
32
elu
ate
fr
om
se
ed
s a
t d
iffe
ren
t pH
v
alu
es.
DE
32
elu
ate
pre
pa
red
fr
om
se
ed
s
tha
t h
ad
bee
n
ge
rmin
ate
d
for
24
h
was
u
sed
. *\,
pre
cip
ita
tio
n
an
d
assa
y
05
pro
tein
s
in
this
ex
tra
ct
was
pe.
ylf
orm
ed
wit
.h
the
sa
me
pro
ce
du
re
as
de
sc
rib
ed
in
T
ab
le E13.
Dat
a sh
own
rep
rese
nt
Am
ersh
am
[3
~]
~~
,
bo
un
d
(prn
~l*
mg
-'
pro
tein
),
I -
I
Inc
ub
ati
o'n
D
E3 2
PH
F.
S..
r
a a
s~
ay
ed
at
a p
rote
in
co
nc
en
tra
tlio
p
of
<0
.2
mg
-ml-
l
- f. Open column gel permeation chromatography
- - - Sephadex G:75 (2"L5x27 and Sephactyl 5-200 (2.5x52.5 cm) - d
were tested for a molecular size-based purification of the GABP.
The binding data obtained with column fractions indicated a -
molecular weight range for the GABP between 50 and 150 kdalton,
supporting the results of Keith - et e. (1981) . ' This method had
poor resolution, low sample loading capacity and slow speed.
use of these column matrices, therefore,-did not seem-to be
practical at &his stage. ,'3
The
Further attempts at GABP purification are described in
Appendix
I - /-
Specific GA bind.ing has been detected in all cucumber '
tissues examined. Thus it is present in 'ungerminated seeds as
well as in GA-responsive and -unresponsive seedling tissues.
However, on. a g'J fresh weight basis, GA-sensitive apical "
hypocotyl sectjons of 7 day old seedlings have the highest GABP _ c
concentration (Table El, Figs. El and E2, and also Keith -- et al.
1982). These results suggest that a tissue's GA sensitivity may
not only be determined by receptor numbers but also by other 6.
- factors, such as receptor activation, gene availability etc.
-
( s e e Michell and Houslay 1986).
~lthough a number of methods were examined for the
purification of GABP frgm cytosol, only a small enhancement of ?
Specific binding was achieved. (NH,),so, precipitation followed
fractionation of cytosol and remova.1 of proteins that carry a
net neutral or postive charge at pH 7 from the GABP. These steps
resulted ih o i ly about 3-fold Purification - of the GABP (Table E 7 ) . Some further purification of this protein using a
hydroxylapatite gr a blu? dextran-agarose column was
demonstrated (Tables E8 and E9). Specific binding was detected
in fractions bound by FPLC chromatofocusing or ion exchange
columns. GABP could also be recovered from precipitates obtained _ - -- - - - _ ----
after lowering the pH of DE32 eluate from pH 7 to pH 6.2 or 5.7. d
However, low recoveries and/or low enhancement of specific
- B i n d b y s i t e &egradatim may be onefacturmniributing ,to
the low degree,of purification achieved. The similar -
chromatographic properties of the numerous proteins in the -
cucumber extracts is another. Thus the GABP coelutes with the
_ majority of the proteins from a chromatofocusing column (Fig. /
~ 8 ) . HPLC - gel f i l t r a t ion~chromatogra rns (Fig. E5) and SDS-PAGE -- - -- ---
of partially purified GABP (~ig. E7) also demonstrate rhe
complexity of these extracts and point tc the expected
difficulties in
purification.
achieving hi(gh yield and high resolution GABP
PART F
Conclusions
The development of an - in vitro assay for GA binding proteins
of GA receptors. In the present investigatiorr~tors
influencing the detection of GABPs were examined. The assay 9
allowed some characterization of the active site of a candidate
receptor in cucumber hypocotyl extracts and,was used for the -
development of a proto for the par 4 ial purification of this , - L-
C protein. Several conclusions can be drawn from this work: Many
nonreceptor entities bind GAS. The reliable detection of GA
receptor-like proteins requires discrimination agaiwt such
' compohents. In Part F I introduced, therefore, measures to
increase the ratio of specific to nonspecific binding. I t was
also deemed necessary to assess binding specificity of candidate
protein fractiois' using a number of ligands of differing - - - in vivo
bioassay activity. - _
The filter assay :s anonequ'ilib-rLium methodan$mLaynot
7 favor optimum GA - GA receptor interaction. Hence it will result /
in an underestimation of the affinity and number of binding
sites in a sample.. %
In spite of these limitations, the - in vitro assay allows the ' -
characterization of the GA binding site of GABPs in the absence
of the effects of metabolism, compartmentati-on, or permeability
barriers which complicate such analysis with -- in vivo assays. In -- Part D it was demonstrated that soluble eABPs kn cucumber- --
extracts bind GAS saturably and exchangeably. The binding
protein shows great-structural specificit-y for ?-lactonic €-I9 -
GAS with a 3 P-hydroxyl and a C-6 carboxyl group. Such GAS are
or D ring of the - ent-gibberellane skeleton or methylation of-the /- - I
C-6 carbo.xy1 result in requction or loss of binding affinity.
~hes; - in vitro specificity data' are generglly suppofted by
published -- in vivo bioassay data on the expected conformation of /-
the active site of the GA keceptor in cucumber. I
+ - 7 - -
d
The GABP is present In 1 ungdrminated seeds as well' as ISrget , *
and nontarget tissues'of cbcumber seedlings (Part El. However, I
on a g - ' fresh weight ba i s extractable GABP activity is highest 2 \
- -
in apical hypocotyls, the tissue which also shows the highest - in
viva sensitivity to GA application. -
Overall, the-GABP studied con.forrns with the characteristics
-expected ot GA receptors. 4 modest degree of purification of
chromatography' followed by chromatography on a hydroxylapatite ? I
or a dye-ligand affinity cqlumn (part E). However, further work I
on this aspect is a prereqdisite to allow elucidation of the
true cellular function of dhis protein. I
PART G
APPENDIX 1 -
1 . I L e v j ~ w af d Molecular Ef fwt3 of G&s Us
Mobilization in Cereal Aleurone as a Model System
P - Research on the molecular basis of GA action has mainly .
focused on substrate mobilization in cerea1,grains. This system
is reviewed in this chapter with- a view to the potential role of d
I
GA receptors. Reviews of these and other morphogenetid and/or - bio~hemical aspects of GA action can be found in Jacobs n - P - -
1
(1'983), Jones ( 1 9 7 3 ) ~ Letham -- et a'l. ( 1 9 7 8 ) ~ and Zeroni and Hall
The most detailed understanding of GA action has been gained
' fr,om the cereal aleurone 'system in which GA can alter the
pattern of RNA and protein synthesis. In cereal grains, GA
released by the embryo controls the -- de novo synthesis and
secretion of several hydrolytic enzymes in cells of the aleurone
layer. Theskhydrola@ses mobilize ehdosperm nutrient reserves - - - -- --
required by the developing seedling.
The most dominant hydrolase - synth d is a-amylase which
8 -
'-23 in barley-comprises a family of 2 groups of high and low p L
isozymes. In addition; GA, stimulates the synthesis of
0-glucanase, acid phosphatase, RNase, DNase, and several
proteases (~ammerton and Ho 1986, Mundy - et al. 1985):~~ J
1
inhibits the synthesis of another seed protein,
a-amylase/subtilisin inhibitor '(Mundy -- et al. 1986). These -- - - - - - - - - - -- - - -
actions of GA are generally inhibited by ABA.
A 3 to 8 h lag-period between the application of GA, t~ --
Ceembr'yonated half Seeds anduthe onset of a-amylase synthesis is
usually reported (eg., Higgins -- et al. 1976, Jacobsen 1983).
However, Muthukrishnan -- et al. (1983a) observed lag times as
short as 1 h. A continuous presence of GA, is required - throughout this lag period. . -
Cell-free translation studies have established that the - - - - --
appearance of 'the GA-induced proteins is preceded by increased
levels of their corresponding & novo synthesized mRNAs. --
Increased transcript stabiliky may also contribute to this - --- - - --
effect. This has been shown using RNA isolated from whole cells,
as.well.as protoplasts and nuclei prepared from oat and barley I
aleurone (~eikman and Jones 1986, Jacobsen et &. 1985, Jacobsen
and Beach 1985, Zwar and ~ooley 1986). while the levels of'
certain mRNAs increase in response to GA treatment, rRNA and . - - - - - - -pppp-- -- - - - - -- - - - - --
total RNA transcripts decrease in barley aleurone (~acob2en and
ch 1985). Muthukrishnan -- et al. ( 1.983a), using cloned
a-amylase DNA probes, detected a-amylase mRNA within 1 h of GA
application. Levels of these-~RNAS increased with increasing GA
concentration. However, rapid mRNR induction, equivalent to that
reported by Theologis et e. ( 1 9 8 5 ) ~ who observed auxin specific RNAs within 15 minutes of auxin application to pea epikotyls,
%
have not been reported.
Several deta-ils about theL e•’fectPof GA n?ran%?lption are
noteworthy: All studies' indicate that a cytoplasmic factor
(receptor?). is required for GA to haire an effect +on gene -
F
ekgression. GA-specific RNA transcripts are always isolated 8' -
-
aftcr whole aleurone cells or their protoplabts have been -- - i
incubated in GA,. Incubation of isolated npcjei in GA, has not
been reported to result in a specific . A similar -- requirement by nuclei for a soluble ic?) factor was
ti noted by Johri and Varner ( 1 968) for 'pea shoo s. Difficulties in a
prep<aring undamaged nuclei could, however, alsk result in this
phcnn~benon, Further support for the presence o GA receptors-_in - i: - - -
aleurone tissue comes from the work of Keith - et g . (1980) who
observed saturable and dpecific binding of [ 1 A, by barley 3" alelurone. This work is discussed in more detail
- --
'Evidence forsthe .Existence - of GA Receptors'.
Differential regulation of the 2 groups ,
isozymes which'are encoded by 2 different
genes located on different chromas.omes h'as been ob efved by 4 ~handlef * - - et al. ( 1 987) and Ho -- et al. (1 987). They observed that
the mRNA for the group with a high isoelecfric poind, (PI) is
induced by GA3 within , 3 to 4 h and reaches a maximum\at. 12 to 16
h &fore declining to low levels at 24 to 48 h. trast, the ',
mRNAs for the low pI isozymes are present at low
even in-the absence of GA3 but increase several-fold w th GA, k treatment. High levels of these mRNAs perList for at leGst 48 h
(Chandler -- et al. 7987). I The k~-rece~tor may; 4 -
with several chromosomal sites.
- - - - -
Work by Muthukrishnan -- et al. (1983b) suggests that in4uction w d
of .-amylase mRNA by GA >equ5res one or moie concomitantly - - - \ --
%
synthesized protein(s), as inhibition of protein syntheSis by,, .
-- - cycloheximide res'ultSFin a drastic decr&< inu-amylase mRNA
levels even when GA is present. No iurther reports on this \
- k-
potential regulator appear to have-been published.
B I + ,
The role of ABA in these pcocesses is not 'clear. ABA $ -
inhibits d~-prom~ted changes in.gene'transcrrption (Jacobsen and
Beach 1985, Zwar and Hooley 1986). Recent work -from the 3 . - - - - - - -- - - - - - a - - - -- - -
r
1aboratories.of Ho and Jacobsen suggests that ABA induces the
synthesis of a ps40tein' that inhibits GA-induced mRNA synthesis
(Chandler -- et al. 1987, Ho -- et al. 1987). Furthermore, ABA may - - - - -
- - - also in•’ luence GA-induced drotein synthesis by having minor
effects on post-transcriptional processes (~iggins -- et al. 1982). - -
ABA action on the GA-.receptor appears to be unlikely. . . - \
In addition to controlling gene transcription, GA may also i
influence the synthesis and/or turnover rates of intracellular '
- - -- - - - -- - -- - -- - p-p-L - - - --
membranes, especially the' ER, and thereby af fect protein
synthesis and secretion. Evidence for such action is, however,
mostly circumstantial. Thus Collins -- et al. (1972) reported
& increased 3 2 ~ incorporation into cytidine triphosphate within 30
min of GA application. The authors suggested, but <id not
demonstrate, an involvement of these nucleotides in membrane 4
biosynthesis. Activation, independent of -- de novo protein <
synthesis, of phosphorylcholine-cytidyl transferase and
\ barley aleurone within 2 h af ter GA treatment (Johnson and Kende
1971). These enzyme's catalyze the incorpor-ation of
phosphatidylcholine (lecithin) into membranes. - In vitro
activation of these enzymes occurs within minutes after GA, - - - A - - - - --
application and is inhibited by ABA (Ben-Tal and Varner 1974).
GA-promoted -increa;es in lecithin incorporation into ER
mehbranes have been reported by Evins and Varner (1971) and have % C
been attributed. to an incyeased turnover rate of membrane
constituents' (Johnson and Kende 1971 1 . These events have been *
reported .to be followed by increases in rough ER ( ~ E R ) and - - - .
polysomes starting 3 h after GA treatment (Jones 1969, Vigil and
Ruddat 1973). The rER has been associated with the synthesis of - secreted proteins such as a-amylase (Jones ahd Jacobsen 1982).
i
Some of the nonsecreted a-amyla5k isozymes may be produced in x
J
the polysornes. However, a di;ect. effect of GA on rER and I
polysome synthesis as suggested ,by Collins -- et a1.h(1972) has
been questioned by the biochemiGal studies of Rodaway and Kende
(1978). Also, Gibson and Paleg il976) observed no GA-induced 1
membranes, and Zwar and Jacobsen ('1 972) detected no significant t
changes in uridine and adenosine incorporation into rRNA due to
GA. Colborne -- et al. (1976) criticized the'work of Jones (1969)
and Vigil and Ruddat (1973) for a lack of control micrographs of 4
aleurones incubated in the absence of GA,. However, their own
ultrastructural study was cha~acterized by a paucity of figures
showing the effect of GA treatment. Nevertheless, Colborne et - al. (1976) did show that exfensive ER development occurs -
--- - A - -- -- --
independent of external GA, application during early h
germinat ion.
- ,
It is not clear whether the observed changes in membrane, Y
are independent events accompanying other metabolic change.
Whether GA directly af fects rER and polysomes and thereby -
regulate post-transcri,ptional processes also remains-
unresolved.
. A direct involvement of membrane 1 in GA action has L L
been suggested by Singh and Paleg ( 1-985j. These authors examined
dwarf wheat which contains a lesion at the Rht 3 gene. The
response of this mutant to GA, as measured in -terms of hydrolase-
production by aleurone and leaf elongation, is retarded (Ho et - al. 1981) . Singh and Paleg ( 1 9 8 5 ) observed that this mutant has -
d' low levels of phosphatidylinosito4, -choline, and -ethanolamine
the aleurone tissue compared to the normal,.GA-responsive,
11 wheat. Low temperature incubation or a 4 h IAA exposure of
the dwarf aleurone-prior to-GA -treatrnmt resulte6 -in7nT;--easea-y-----p
synthesis of these phospholipids and an enhanced response to GA, -.
(Singh_.and Paleg 1986 ) . The authors suggested that the
correlation between membrane lipid composition and GA,-indyced
a-amylase synthesis -points to membranes as determinants of GA
sensitivity and sites of GA receptors. However, the authors did
not examine the possibility that differences in gene , -
availability, transcription, translation or ER membrane
proliferation exist between the dwarf and t%ll cultivars. Such - - --
differences could affect the ability of the dwarf to respond to
P plications. In this context, it may be worth noting that -
on intrinsic membrane proteins - -- (carruthers - - - - -- - and Melchior 1986).
The literature oh the aleurone system clearly establishes
that GAS can influence gene expression resulting in a specific - -
hormonal response. A soluble car extranuclear receptor appears to
be involved in.the.mechanism of GA action. Such a receptor
remains to be purified and characterized. It is likely that the - - mL -
B
GA receptor interacts with a number of targets, such a s
different regulators of gene expression. The role of GAS in
controlling cellular membrane organization,remains unclear. >
PART H
APPENDIX 2
The
ater rials and ~kthods.' P a r t E,
1. ~xamination of Other In Vitr0 Assays for GABP
methods for separation of bound from free ligand were examined
with the hope of
for detection of
1. Sephadex - G-25
developing an alternative, sensitive technique
minicolumn assay
a. Materials and d+
Methods
The assay procedure followed that described by Tuszynski - et
al. (1980). Sephadex G-25 (Pharmacia) was swollen in Buffer B -
-packed under gentle suction into' 1 ml pipette tips 4
(#230-1196 Y3K, Evergreen ~cientivc, Los Angeles C A ) which had
a small glass fiber plug at t3e outlet. These tips were chosen '
as'their length exceeded that of other designs and allowed 4 - - - -- - - - - - - - - -- - - - - - - - -- - - --
complete separation.~•’ a 100 MI mixture of Blue Dextran (eluted)
and phenol red (retained) under conditions used for the assay.
The minicolumns were fitte'd onto 10 x'130 mm test tubes and 9 .
/
excess buffer was removed from the columns by centrifugation at
2009, 2 min.
I - %
Aliquots of incubation mixture were assayed by loading 50 or
100 ~1 aliquots onto the min~columns at 2 C. he columns were
then centrifuged at 2009, 2 min. Samples of the eluate were then -- - - -- - - -
collecte'd and radioactivity measured as described previously. -
* . H e s u l t v 2
he resuTts from h e G-25 m i n i c o l G i n p ~ y were inc'onclusive
able HI). Receptor-type inding appears t6 be detectable for k 190 ~1 samples of G-25 eluate. However, data variation was very
high; some data for replicat-es differed by one order of -
magnitude. These results have, therefore, questionable validity. ,
, The method was labor intensive and had many sources of - - - - -
I
variability. It was therefore abandoned.
2. Nitrocellulose filter assay
a. Materials and Methods
This assay is based on the ability of nitroc llulose r membranes to bind proteins and with them bound ligands. The
procedure of Inoue -- et al. (19831, w'ho used-cellulose ester
membranes for the_assay- ~•’_thyroid-receptors w a s
Membranes (type HAWP,.0.45 ~,,.2.5 cm diameter, Millipore 4
corporation) were soaked and then rinsed with 0.5 ml Buffer C
under suction on a filtration manifold. Fifty u1 samples of
inc;bation.mixtures, prepared as described previously, were then
applied. This was followed by a wash with Buffer C,volumes as Y -r -
shown in '~esults'. Membrane-bound radioactivity was measured by
scintillation counting a$ described previously.
--'fabe-H1.ssay of GA b i n d i ~ r c ~ ~ € ~ G = 2 5 e l u d t e using t h e Sephadex' G-25 minicolumn assay . -
-- Data rep resen t average cpm e l u t e d from columns. G - 2 5 e l u a t e . was assayed a t a p r o t e i n L c o h c e n t r a t T ~ i i of 3 . 9 mg-ml-1.
Incubat ion Sample Buffer G - 2 5 volume B e l u a t e
On me basis d t ~ r e = j t r - s h o W n in Figure A,, a 120 ml I
wash volume was ch6wn to reqove unbound [ 3 ~ ] ~ ~ , from + A.
I
nitrocellulose filtkrs. In a further experiment with these I
membranes the effect of sample volume on 1 3 ~ ] G ~ , binding in the
, absence and presence of an excess'of GA, was measured. Column *
buffer and G-25 eluate were used in the incubations. The , - - - - -
presence or absence of cucumber extract did not greatly affect
binding data over the volumes assayed (Fig.- H2). Thus although
total binding in the presence of G-25 eluate was about twice as
high as' in its absence, specific binding vztues did not differ
significantly from background binding. The results suggested
that under the conditions used vexy little GABP is bound by the
membranes. This assay was therefore hot pursued any further.
Materials and Methods
Bruns -- et al. (1983) have shown that acidic proteins (PI less
than 7 ) -are stronq&y'bound by the polycationic polymer *"* - 2,
polyethyleneimine ('PEI ) . ~he~''h3ve used PEI coated glass fiber I '
filters successfully for the assay of a number of animal ,
receptor proteins. Their procedure was followed for.te,sting its .
BP assays. Whatman GF/B glass fiber filters (2.5 cm ; > .
--- - --- -- diameter) were soaked in 0.3% (v/v) PEI in water, pH 10, for 2 h
before use and were placed on a filtration manifold without %
127
Figure HI. Effect of wash volume on [ 3 ~ ] ~ ~ , binding to t nitrocellulose filter paper. Fifty p1 aliquots of incubation mixture containing 64 nM E 3 ~ ] G ~ , in Buffer B were loaded onto nitrocellulose filters. Af te diff Tota
r 1 min, suction was applied and filters erent volumes of Buffer C. 1 cpm applied per filter = 153,000 cpm.
washed with
Figure ~ 2 . Effect of sample volume and presence and absence of i
G-25 eluate on [ 3 ~ ] G A u binding to nitrocellulose membranes, Samples of incubation mixtures containing 50 nM [ 3 H ] G ~ u in the absence and presence of 5 pM GAu were applied to filter disks yielding total and nonspecific binding values, respectively. 'Spec-ific' binding was determined by
2 .
subtracting nonspecific from total binding.
C I - i
@-a 50 nM [ 3 ~ ] ~ ~ 4 + G-25 Eluate - - \
r M- - 'Specific ' With G-25 Eluate
Sample Volume (ml) - - -
- * m I
- 50 nM [ 3 ~ ] ~ ~ 4 + Buffer i ) - - - - - - - - -
&--- 4 ' Specific ' With Buffer
washing. Duplicate 50 11 samples o,f Incubations of 50- nM ['HIGA, '
in buffer B or G-25 eluate, were loaded onto the 'filters. - -
Various volumes of Buffer C were used as indicated i'h 'Results'
to remove unbound radiolabel. Bound radioactivity was measured
by scintillation counting as described previously.
b. Results
PEI-treated glass fiber
significant amounts of GABP
4. ultrafiltration membrane
a. Materials and Methods
1 This method is based on
filters also did not bind - - -
(~i'g. H3).
assay
an approach used by Venis ( 1 9 8 5 ) for
thp assay of auxin binding proteins. Separation of bound from
free hormone is achieved by passing the incubation mixture I
FteLeptor-bound hormone is expected to be retained by the I I
mebbrane while unbound GA is washed through. Discs (2.4 cm ' I
diameter) of the 2 types of ultrafiltration membranes, purchased
from Amicon Canada Ltd., Oakville ON, were tested. PM20 ( ~ o t
2960) and YM1O (Lot 2800) were used. Acc rdinq to the supplier, 9 thd PM20 membrane is made from an inert, nonionic polymer which
0 tends to bind hydrophobic molecules and has a .molecular weight
-
cut-off for globular proteins of 20 kdalton. Compared to the -- -- - t- --
PM29 the YMlO membrane is more hydrophilic and has low,protein
bindipg properties. It has a molecular weight cdt-of f of 10
Figure H3. Effect of wash volume on [ 3 ~ ] G ~ , ' b i n d i n g to FEI-treated glass fiber filters in the absence and presence of G-25 eluate. Fifty ~1 aliquots o f incubation mixture were loaded onto filters and immediately washed with the volumes indicated. Total cpm applied to each cpm.
---
of Buffer C
G-25 Eluate - -
Buffer
Wash Volume (mi) .
- FOF t k a f l ~ i ) ~ , FQQ +d =m&= QT *htior-&urcs w c r ~
loaded onto presoaked membrane discs'undere suction. The effect
of different wash volumes as well ai competitors was tested.
Membrane-bound radioactivity was measured by scintillation
counting as described previously. @
, .
b. Results
The PM20 ultrafiltration membranes seem to have a greater 4
protein binding capacity and better flow properties than the
YMlO filters. A wash volume of 2 ml appeared to be suffi dient 3 . *.
. tor the removal of unbound radiolabel (Fig. H4). This wash ,"
required about 1.5 min and was used for a-follow-up experiment b-
in which binding in the presence and absence of different
competitors was assayed. Some GABP appears to be retained by the
data in the presence and absence' of G-25 eluate was quite small ,
(compare with DEAE cellulose filters, Table C 2 ) . This
ultrafiltration assay did, therefore, not appear very promising. .
.Figure H4. Effect of wash volume on [ 3 ~ ] G A , binding to ultrafiltration membranes in the presence and absence of G-25 eluate.
- - Fifty nM [ 3 ~ ] ~ ~ , was incubated in the presence and absence of G-25 eluate. Aliquots ( 1 0 0 ~ 1 ) were loaded onto s
ultrafiltration membranes under suction and bound i
radioactivity determined. I). Total cpm appIIedperfiIre r = '250, om-c pm: With- --
membranes, washhng times for volumes greater than 0;5 ml , exceeded 10 min and thus were not assayed.
I I I I
I
I ) .
G-25 Eluate
Buffer
G-25 Eluate Buffer
Wash Volume (mi)
\ Taele H3. C i-indjng of [ . - l G A i * Q PM2Q - - - -
3~
u l t r a f i m n membrane d i s c s i n absence and p G-25 e l u a t e .
- % & - & - m m k ~ m +ses were r M - w i t h 0.5 d Aliquots of Incubat ion mixture were passed t h r
fl s u c t l s n and f i l t e r s washed with 2 m l Buffer C t o remove unbound r a d i o a c t i v i t y . Data represent cpm bound per 100 p 1 sample. G-25 e l u a t e was assayed a t a p ro te in concent'ration of 3 . 6 mg-ml- ' .
'*
-* - ,/ Incubat ion Buffer G - 2 5 (r
B eluate --
50- 'n~ ["]GA, 500 690 + 5 pM GA, 350 490
\ + 5 p M GA, 390 480 + 50 p M GA,ME 3 2 0 5 7 0 i'
- 1 1 . Further Attempts at GABP Purification
t
a. Materials and Methods i
. 0
Two types of ultrafiltration devices purchased from ~rnicon
' Canada Ltd, Oakville, ON were tested for increasing the purity
and/or con'centration of the GABP. 'Centriflo' membrane cones, - - - -
d' - CF25 amd CF50A, ( 2 5 and 50 kdalton cut-off, respectively) were
used in a centrifugesat 8009. Diaflo ultrafilters PMlO and
XMlOOA (10 and 100.kdalton cut-off, respectively) were used in a
model 8050 stirred ulrraf Cltration cell on ice.
b. Results
~eceptor-type binding.was retained by both 25 and 50 kdalfon
membranes, however, specific binding was not impr6ved (Table
- K-3); Displacement-a•’ { % ] G A , ~ ~ - G & M E was--iRcreaa&irrkhe--c-cme- -
fractions. This may reflect some changes in the configuration of
the GA binding site of the-GABP. No protein and, no GABP-type
binding was observed in the fractions not retained by the
filters. Slow speed, liiited capacity, and variability in flow
rates between cones led to the discontinuation of their use.
10 kdalton stirred cdll filters retained GABP. Total [IHIGA, I
binding increased, data quality was however poor (Table H4).
Results obtained with 1QO kdaltan stirred cell ultra f a3 tzati9t-i ' /
membranes were also inconclusive.
removed from ~ ~ 3 2 e l u a ' t e at 7,0009, 5 min. ingo the cones with different
molecular weight cutoff. The canes were *repeatedly* centrifuged at 8 0 0 9 and washed with buffer B.
. .
-
~ncubabion DE32 eluate >25 Kdal > 5 0 ~ d a l
- -
,
Table H4. ~indinq of [ 3 ~ ] ~ ~ 4 to DE32 eluate retained by 10 Rdal ultrafiltration membranes. Particulates wereremoved Ero63~32 eluate at 70009, 5 mine , ,
The supernatant was loaded into'a stirred ultrafiltration cel1,containing a .I0 kdal ultrafiltration membrane. The sample was concentrated under pressure and repeatedly washed. Data .represent pmol [ 3 ~ ] ~ ~ , bound-mg-I protein.
Incubation DE32 eluatea >10 ~ d a l . ~ >10 ~ d a l ~
50 nM 1 3 ~ ] G ~ , 0.23 0.41 0.13 + 5 DM GA, 0.10 , 0.27 0.05- + 5 pM. GA3 . 0.10 0.38 0.09 +50 DM GA,ME 0.19 0.35 e 0.14
aassayed at a protein concentration of 1.2 mgoml-I bassayed at a protein concentration of 3.7 rngJrnl-I
ion membranes had slow.flow rates All types-_ofulArafiltrat
with the relatively crude protein solutions 'used. In the stirred - - - - - - -- - - -
cell .system precipitates would form
proteins.
2. Nondissociatinq - PAGE
J' a. Materials and Methods
- -,
suggesting denaturation of
Preparativ?, nondissociating PAGE was performed adopting the
method of ~brambvitz -- et al. ( 1 9 8 4 ) . A 1 ~ 1 2 ~ 0 . 3 c m stacking gel
( 3 % T I 2 .7% C in 0 . 0 3 8 mM Tris-HC1, pH 6 . 5 ) was cast on a *
, pre-electrophoresed separating gel ( 1 2 . 3 ~ 1 2 ~ 0 . 5 cm, 6% TI 2.7% -
C ) in Tris-glycine electrode buffer ( 0 . 1 9 2 M glycine, 0 . 0 5 M
Tris, pH 8 . 6 ) . This buffer was also used in the upper and lower
buffer chambers and maintairied at 1 C.
Lyophilized DE32 eluate was resuspended in- buffer 8 , washed '
- - - -- - -- - - - - - - -- - -- - - - - - - - -- - - - -- - - -- P -
and reduced in volume on-a C 5 0 A ultr.afiltrqtion cone. A portion
of the > 5 0 kdalton fraction was retained for assay, the rest
( 2 . 6 m-1, 6 mg protein) was diluted with 0.6 ml sample treatment
buffer and loaded onto,,the stacking gel.
A current of 2 3 0 mA ( 9 0 0 V) was applied for 15 min followed
by 70 rnA'constant current for 30 rnin when the brom%enol blue
(BPB)' dye front had migrated 2 . 5 cm into the separating gel. The .
top 3.5 cm of the-slab gel were divided into 5 strips which were - - - - - - - - - - -- -p -
inserted into 5 0 kdalton cut-off dialysis bags (spectrapore 6,
3.4 cm, Spectrum ~edical Industries, Inc., Los'Angeles C A ) . -
~ l s o , a 1.5 cm wide strip in front of the BPB 'front 'was
collected for '~chtrol'. ~1ect;oelution of proteins from the gel *
~ t r i p S a T p T f o r m X w i t - h ~ t 5 1 7 T c o n s t a n t voltage at 2 ' ~ •’&r 2 h.
Gel fragments-were remov d by centrifugation. The supernatant a was washed with buffer B a h concentrated in a CFSOA - ultrafiltration cone for assay.
b. Results
-
The feasibility of recovering GABP from extracts -
fractionated on nondenaturing polyacrylamide gels was tested.
DE32 eluate was loaded on preparative slab gels and
electrophoresed 4 cm into the-slab. -Recovery of total proteins
by electroelution followed by ultrafiltration was pooe (25%).
Table H5 shows that no GABP activity was recovered by this
procedure. A follow-up experiment was performed. This indicated , 1 '
that this loss could have been due to GABP denaturation during
ex pasure-Lo the ele&aphnr e s F s - b l l f ~ Tahk -& s h w ~ t h a t - - ---
during the minimum time required to perform preparative*
electrophoresis, 5 h, considerable loss of specific binding
ocdurs. The electrophores s buffer used was at pH 8.6 but has an I actual operating pH near 10 when used during electrophoresis. A
number of other buffer systems which, according to ~ o v i n et al.. -- . ( 1 9 7 8 ) are supposed to operate Gear pH 7, were tested but none
gave satisfactory protein 7
mobility and banding patterns.
-
7FZhla2TECiKding of [3H10A. to UBJ2 eluate besfore and after pr parative PAGE. DE32 eluate was assayed before and after preparative, ' electrophoresis. A sample that was electroeluted from a 1.5 cm wide gel strip in front of the bromophenol blue dye front was used as a control. Data represent [ 3 ~ ] ~ ~ , . bound (pmol-mg-I pro.tein), exce5t for control (pmol-rn1-l).
Incubat ion' control. D. ,32 eluate before PAGE after PAGE
- --
I S ~ ] ~ ~ , displacement given in parenthesis.
f I
Incubation cantrol buffer B PAGE buffer
50 nM L 3 ~ ] G ~ , 0.40 0.51 0.35 \
+ 5 UM GAQ 0 . 1 6 ( 5 9 ) 0.20 ( 6 0 ) 0.26 ( 2 8 ) + 5 BM G A 3 0.29 (281 0.25 (51-) 0.-28 ( 2 2 ) - +50 pM GA,ME 0.-34 ( 1 3 ) 0.35 ( 3 3 ) 0.32 ( 1 0 )
PART I
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r
Nasser Y a l p a n i Dept of Bio log ica l S c i e n c e s Simon F ~ a s e r University,
AG)J/).'F/F 2 4 July 1 9 P 7 ,
Y a l p a n i
W i t h reference to your atta&ed r w e s t to reproduce material from a Pergamon =
Journat, we herewith grant yQu permission to do so: f
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, Nasser Yalpani Dept. of Biological Sciences. Simpn Fraser University
' Burnaby, B.C., Canada V5A 1S6
July 16, 1987
I would like to get your permission to use a figure tha t appeared in . 'Ph.ytachernistr~' for a literatwe review as part of my Ph. D. thesis. The article is: Serebryakov. P.E., V.N. '~gnistikova, L.M. Suslova 1964. Growth promoting activity of &me selectively modified gibberellins. Phytochemistry 23: 1847-1 854. The figure I -would like to use is Figure 2 on page 1852. The diagram w0ul.d appear unmodified, exeept Fig. 2a would appear above Fig. 2b. A credit to the - original authors would of course be given:
Your rapid response will be appreciated. . . )
J
Sincerely your%.
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