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INTAACELLULAR FOtATE OF HlI4M
F18RClllAST S
Soudabetl A. Kutlan1
A thesh sut.1 tted to the Facu1~ of Gr.duate~ud1es and Rase.reh in parttal fulf111.,.t of t .. requ1re.nts
for the degree of Master of Science. ,
Dlpart.nt of Physio1ogy.
Mc6tll Untve"tty.
Mont .... 1. P.Q .• canada. @ August. 1984.
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-\!i\!&~1iJ.;U4M' Ji4Jl .... ~~.,. ";;;U",,*,,*"'i U.$.14 JIU _1711&4 ___ #OUMI'" te. Il00 ..... _ _ __ ,_ .... _ "El il: • 1; RtM_J._ -
lBSftlCT
Tbe eodogenous leyels of yarious folate .oD09la~a.!te
:::oapounds in :::ultare:1 baaan fibroblasts "ere deterained
Il S iog big h perf oraance l iquid chroaa tograph y for the
separation of folat. aoooqlQ~aaate. Endoqeoous folates "eee
conyerted to aonoqlutaaate foras using the conjuqase eaz,ae
present in ra t sarua and in cu bation "as carried out at pB
6.5. Tbe use of tbis procedure ainiaized folate coenz,ae
lnterconyersioD 1urin9 processin g_ Osing aet banol for
precipitation of protein instead of beat aioiai zed the
~e9radst1on of labile folates. RecOyerf of all folates
eK:::ept 10-C80 84PteGlu using tbts procedure "as aore thao
'JOI.
Disruptioo of cells by boiliog appeared ta cause lass
post- •• traction cbanqes of cell folates tkan did treezing
and tbaving or sooicatioD.
oalng beat to release _dogeoOQ8 folate, coojll'jilse
teeata.nt vith rilt seru at pB 6 .. 5 and precipitation of
protain vitb letbaool, aore than half of tbe iotracellular
folat e of nor _1 f iJu;oblasts io collfl_nt qro.th vas 5-C113
84Pt&11u, and 10-C80 H4PteGlu and a.,teGlu co.pri •• d 2i and
6 percent of ead()()enOQs folate r_pect.i •• lr.
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·~~~."AI~.ftlMi~jl Z$IJ!8!Ul tt;'1iI0IS"W'AAlOkai_oas*_, ... _., "' ..... " _ h
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RESUltE
Les niweaux endogene8 cles diwers co. posés de la folate
aooogluta.ate dans les fibroblastes de cellules bu.ailles
cultiyees ont ~té deter.ioes par l' e.ploi cle la
cbro.atograph ie liquide a ha ute pre s8ion (HPLC) pour la
separation de la folate .onogluta.ate.
Les folates endagenés étaient transtora.r sour fora.
aOllog lu ta.ate duran t une incubation a pH 6.5 a Yec en zy.e
conjuga se présente dans le seru. cl. rat. Cette tecbnique
.ini.lsai t la CODye rs10n de la folate clue ut le procéde. L'
a.ploi clu aéthanol pour la précipitation des proteines
p 1l1tot que la chaleur .i Di ai sait la degrada tion :les fols tas
1 nstables. La récu pérat ion de tous les folates acept 1.
lo-cao B16PteGlu par cett e technique était pl us de 901.
La scission des cellules sui te a une ebulli tion
pIOwoquait .oios cle cJlange •• nts aprés ex tract ion clea
cellules folate que le qel, le cl8qel et le déae.bra.eat.
L' utilisation de la cbaleur pour libérer la fol~te
endogéne , le traite .. nt a pB 6.5 a 1 Gnz,.e Ayee le •• ru.
de rat et la précipitation des proteiDes aYec le .éthanol
oot par.is cle déter.iDer qae pllls cle la .01 tié de la folata
intracellulaire des fibroDlastes nor_ a)[ en plaa .. conf la_a;::.
était 1. S-CB3 a'Pte~lll, alors 'la_ 1. 10-CIO a.'teGlu et gu.
le I14Pteala co.posaient 29 et 6 poGJ:Ca.t r .. pectiy __ t.
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Tl BEL OF COlT BIT 5
CHAPrER 1 rlTBODOCTIOI
1.1 Folate Structure and Blocbe.istry •••••••• 1
1.2 Polate !azy.a •••••••••••••••••••••••••••• 8
1.3 Polypolygluta .. te Sjntbesis ••••••••••••• 27
1." fI ••• all. an Polate Transport •••••••••••••• J 1
1. 5 Eni OCJiI DOUS Pola te in fI .... 1ian Cell. •••• ]5
CHAPTES 2
':1I1PTER J
CHAPTB. Il
CHAPTal 5
CHAPTD 6
fliCJb Perforaan ce Liq uid C broaatogra pb,
TacbniqIl8 •••••••••••••••••••••.•••••••••••• 38
Tbe Parpose of Preaent Stady ••••••••••••••• 'l fil"
flaterials aad .. t.ods •••••••••••••••••••••• 115
a .. alts ...•...•••••••••..•••••.••••....•.•• 55
Diac .. sioa •••••••••••••••••••••••••••••••• 18
8I8LIOGI1PBI •••••••••••••••••••••••••••••••••••••••• 85
111
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Dedicated to:
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.J bQsbaDd, "ho ù"aJs iDspired .e to
achie.e ., goals.
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ICKIOVLB DGBIIDTS
',lrst l woœld like to eKpress.y siocere appreciati~n
and tbanks, to ., saper.isar Prof. B.A. Cooper for Ilis
,JQ.l.d~nce, encoara'J.ent and patience duriog tbis res.arcb.
l IIoold like~to tbank PIs. sally Loe-Shing for tea=blQ~
.e the tech.n ical 5k1.115, an d for ber ad. ice and belp IIi tb
tbe .1crobiological assay.
l "ould like to tbanJt Dr. Oayid 80senblatt liDO
proY1.ded the cell lioa œs.d in tbis stœ«ty and Dr. Rob .. rt
ftaclenzie for Bis belpfull suggestions tbroU')hoat tois ":t
res_arch.
r IIould also lite to tbank "s. Dorli Itoifel for
rl83istlog .e "itb t,pioC] and PIs. Deoise Besuliel1 far
praparation of tbe Praodl abstract for the th.sis, and !la.
catby Grauer for ber assistance IIith tbe celi c1llture.
1 a. 'lrat.fal to the "edical aes.arch Council of )
~4nad3 for grants proYided to De. cooper tbrou'Jàout tàis
st",dy.
Word caD ~ardlJ azpre~s., siocere appr.ciatioo to .J 1
.otber for her loy., e.courage .. nt, support and patia.ce
tàrOUgAout ., lifa.
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w~_ ... ;:." J.qll';ICU\\tl1JlUU'~ '_4. "_._~"I,~ .. III : M4tI.1'''''.''''" ........... _ .. __ ......... (~"" ........ " .... , ............ ..,MMlM ... J .... 4)i! ... 41 ... Ii4A..."r#lhŒ.1tj!.1:1l.-lblfi,..~r '~---
C81PT~ 1
1.1 P~late structore and biocb .. istry:
At tbe beCJinniD9 of tais c8lltury a series Qf pigaeJlts
If er e l.Solated froa tbe v iDCJs of the botte rf l" dll of vrli=A
wera foand to baYe tba sue basic strucrt-are. lIienland (1)
Ilsed the tera' Pteridine' for the bicyclie nitrogen ring
s,ste. of tbese piqaeuts.
1 Du.ber Qf studies on nutrition&! factors in Mn !ed
t:> tbe diseoyer, e>f a substance containiDq the Pteriiiae
str~tore, vbicb has b •• n
intr~eellQlar aetabolisa.
found to pla, a ka, role 1D
11115(2) reportad tbat eKtra::ts
of lltolfsed ,east or li Yer vere effectiYe in tbe tr_taent
:> tropical aacroc,tic an.aia. It vas lat~r shovn t8at
3Dkeys aaintained 00 di8ts siailar to tho. associated vita
huaan tropical aa::rocytic aneaia &lso de.eloped an aneaia
vhich responded to the adainiatration of ,east or liyer
fi Ktracts (3,4). A li yer factor vas f ollnd to he an
eS:Jantial Blltrient for laetic aeld Daet.ria (5, 6' a.~ to
pre.ant aacroertie an .. 1. in eb1eks (1,8).
The Daaa 'folie acid' vas first proposed b, !lite.ell
(9) et al lD 19.0 for the SUbstance vb1ch tbe, had isolated
fro. spinach snd vbicla vas a .etrit ional factor for
Streptococe. fa.cali.. It appr3Kiaatly tla. s ••• ti.. a
fact()r st1 •• 1atiJlCJ growtla of Lac:toAlacUlus c ... i, .. &s
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1 1 f ! {
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1!!ItlI.,IJ!II!.IiJlitillllJ*l.UII:ltn_._.IJI' IIln.!_I!II!$ .... """ .... ~". __ .,.a .... ' .......... ' ... __ • __ • _._._"_ •• _"._,_ .. _____ ~ . _ _ _ ~ _ • __ • _. _. ____ ........ _ •• ,..,p_ .... , .... i4I11J1!I-
isolated fro.liyer(10,11,12). ln 19_6 &Dgier e1: al.(12.
pllrified factors isolated fro. liftr, yeas1:, and spins:h
tb3t vere effectiye in the tr.at.ent of .acrocytic aneaia.
Purification of tbe factors responsible for tbe aboye
nutritional effects reyea~ed that the basic structure ;,f
these factors vas identieal aDd included t he structure 1-(
11- (2-a.ino- .... hydroxyl-6-p1:eridine' .ethyl} a.ino] beDz;,yl
~llltaa1e aeid. Osually pt.ridine refers to 2-a.ino-~-
bydroz, pterin ring and ~erin is Qsed to aIl co.pounds
cOlitaiDinq the pterrin.
'olie acid is pteddine liDked by a .ethylene bridge
to p--a.inobenzoic aeid group, vhich i5 attached throu~b a
pept.1.de bond to a gluta.ie acid .oleca~e(Pigure 1).
rolie acid 15 poorly soluble in va ter and iD organ ie
solyents. The 2-a.1no and lI-hydroxy groops haye "n
insolublizing affect. lpparaotly bydrogen bonding batveen
pterriA._olecales ls reioforced by h1gh dipole .caents (13 ••
The qre.a1:er solabilit.y of the di-sodio. salt of folie acid
thsn of tA. acid apparently is due to the effect of
iODuatioa of the glota.aie acid residue (14 ••
ptarins are relatiyely resistant to oaidation bat
heatlag vith chlorate at acid pH degrades the ring sfste.
.ith 1:b. for.a1:ioD of ~uaDidlo. fro. the 2-a_ioo pyriaidi~8
pottioD of the aolecole. Lite .ost pteroyl coapounds, folle
acid 15 stable to ùJtall QJlder aDaarabic conditions, bot
~lkaline llfdrolfsis aDder aerobic coacJitioJlS cl .. Y8S the
2
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,....', imaM MU aM tS.ilXllIaM;;:;_,""..,. __ ... ""'.z: ... __ lIII'i __ "''''' __ '"" .... ' ... ,,,""'w""" ...... " ... LI!lIlI ..... n ..... s'""'~""', ......... ,_,_, __ ~._. " __ .... \ .'1.32 1 d!4!\tlll!lN
1
<-
c
~
si~e chain to yield
ptarin-6-earboxyli= acid
p-aaino benzoylglutallic acid and
(15). Aerobic bydrolysis in aeid
,ields 6-.et by Ipter r in (15).
Folie acid is only active as a coenzy.e wben it is in
a reduced for.. F:>lic acid and .any:lf its derivitives ean
easiIy be reduced by a variety of agents to the
corresponding dihydro or tetrahydro folate. H2PteGlu and
B4PteGIu (16) are rapidly o~idized by air. The lIain pr~duct
:l f B4PteGlu o~idat ion is p- aainobenzy 1-g luta.ic acid (1 G, 17
) and a nu.ber of pterins. Oxidative degradation of reduced
f:>lates is greatly retarded by tbiol and a s::orbate.
One-carbon units can substitute on the 85 or N10
post tions of El4 PteG lu or lia y for. a brid ge betveen the YS
and 110 positions. Substitution at the MS position
increases the stability of reduced folate. 5-CH 0 H4 PteGlu
and 5-for.ylpterrin are stable to oxygen in neutral or
• ildl, alkaline solution. It aay be concluded tbat in
reduced pterrina and folate derivatives, a for .. yl group at
1-5 stabilizes the tetrahJdropyrazine ring to oxidatlon.
forayi
PteGlu
10-cUO H4Pt.eGI u ls 1 •
also labile and t~ad~ly loses its .~
group in the presence of alkali, •
foralng 10-:00
(18) • 5- foraiaino H4PteGlu i5 a
interaediate in the degradation of histidine in certaid
cells (19) • 5-lIUCB H"PteGI u exhibits tlltravio1et and
infrared absorption spectra, siailar to those of 5-CUO
ft "p teGl u and like 5-CHO B"pteGlll ls unaffeq.ted bJ
3
.r f l
? ;
dt.aspherlC oXY'len( 20) •
Another If')-substltuted derivatiye of H4PteGlu, 5-':fiJ
H4PteGlu, WhlCh lS an iDteraedlate in the biosynthesis :lf
lIethlvDlnt:!, is readily olidlzed at neutral pH ln the abs81l::e
::>t redaclnq a..j~nts. The deqradatlon prodact of 5-:HJ
l:i4PteGlu is c, - .et h y l dibydrof olate( 21) • ')-.eto yi
J1bydrofolate oldte can be redace,l back ta .ethyl H~PteJlu
by nydroqen ln the prec;ence af platlDu. (22), ascorbl.c aCld
(2J) CH thial (24) 1.5 lIore stable thJ.D
H 4 P te'; lut 0 0 Xl. d a t l,ln , par t l CU l ci [ l'f 1. n the pr ~sence .H
dJ::::ass foraaldehy:L:l "hlcn p[~VeDts dissoc1at lOD to H4pte-ilu
(251 • 5-aethyldlhydrofolate .ay aiso be oxidi zed t::> a
structLlr~ lDvolvlD..j rearrangaent ::>t r1nqs.
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- ~r-~~ ......... 'l!"-. ..... _> - ~- ... 14 .-l"!1 --' - .. ~ ..... --, , -." ~ -~
~ _ .. _ - r _ - - - •
, 1 il ~ l
1 i
1 '1 ~ <
~
i \li
~ 1
/' 1
J 0 HN CO-NH-CH-COOH
1'0 1
• • J
~
CH2 iH2 j
1 Q
CH2
( 1 COOH
H2N
" "-
figure 1. Structure of folic acid
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TlBEL l
AB BREV lA TIOIIS
Folic Acid PteGI u
Tatx:ahydrofollC ACl.d
Dlhydcofollc ACld H2Pte:;lu
5-For.yltetcahydcofoll.C lcid 5-CBO Hq pteGbl
')-"'eth yItetr- ah ydrofoll.c lCl.d 5-CB3 BqPt8GLl
10- For.ylte t rab ydrotoi i c lcid 10-CHO HQPteGlu
10-Porayldlb ydrofoll.c lcid 1 O-CHO B2Pte~111
5, 10-fletheny Itetrahydrofolic lcid 5,10-CH=H4Pte~111
5,10- flet hyle ntet r ahydrofollc lc id 5, 10-C82- H4PteGlu
5-ForaiaiDot: et:rèlh ydro folie lcid 5-CBIB-B4PteGlu
6
GiYC1Daa1da Bl.bonacleotl.d
f'oraylgl yC1na al.d Bi bonucleot ld
5-<1. ino- ij-L. i dazole car bo xaa ide
Rl.booucieoti1
5-for.iaa1nide-ij-iaidazole
CarboJ:aaide li iboDllcleotid
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PlBGia
:J18
FGAB
lIC1R
f'UCAB
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1.2 Palate enzy.es:
Tbe coenzyae foras of folate are required in the
iyothesis of purines and thyaidylate, and in the .etabolisa
of certain a.lna acids. lIa •• allan calls haYe seYeral enz,aes
tllat 8stabollze pcs-foraed folate. but the, are anable ta
sfothesize falate de noyo. iithin the celi, falate loS
c aDyerted
j ec lyat i yes,
Bnzyaatically
vhich fllnctiaD
inta il y il{' let y at tala te
as coenz yaes in cl nllaber of
rtH::tl.OnS in aaa.alloan calls. Enzyaes shawn to utill.ze
falate coenzy.es IIill be discused here.
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Polate intercon~ersions
o l.h,drofolate redllct ase:
o ih, drofolate redactase ca tal rze 5 the nOPH -depen:ient
reduction of H2PteGl a to B"PteGla (reaction 1). lt a sl.:>.,ar
rate, Dibrdrofolate re:luctase vill also rednce folie acid to
R2PteGlu (26) (reaction 2). The enzr.e i8 present in 111
aaaaalian cells.
H2PteGlu + nOPB + H ::;;<=====" B" Pt eGlll + Il OP
(Re ac ti an 1)
PteGlu + NAOP H • 8 :;::'<=:=:==~> H2PteGlll + HlDP
(Be action 2)
The X-ray structure :li the binary co.pleI vith
aethotreIate ('1-182, 10 CH3 PteGla) as folate binder shOlled
'i central light-stranded beta pleated sheet of the
polypeptide backbone vith tvo helices, vith their aIis
runlllng alaost parallel to the nearby beta strands (&2).
The aet hotrea:ate ao lecllie i5 bound in a 15 1 deep cavity
vith the pteridine rinIJ buried in a priaarilr hydrophobie
pocket close to tbe binding site of • &DPH.! he aro.atie ring
~f the p-aainobenzorl portion of aethotrezate residues sits
in a second hydrophobie poeket. lt least 13 a.lno acid
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residlles &re inYoly.d in tbe bindiDg of aethotrezata to
dihydrofolatQ reduetase (62,.
Dib,drofol,ste reductase is iDhibited reyersiblJ :>r
irr.yersibl, by seYeral type of agents including
aet hotr e:a:at e, aainopterin, (16-IH2 P teG 111' and oth er II-aa1.o:>
lnslogues of folie acid (26, and by a ,ariety of other
cheaical co.pooods. Tbe enzyae bas a aolecular veigbt of
l.1000-lll000 vben isolated fro. cbicken liYer (14'. lcti'ity
ot the enzy.e Increases in tbe presence of certain agents
In:=luding ure!! , sal ts, and guan idin at • hieh disrllpt
.acroaolecular structure, and aqents like aercurials vbich
relct vith the thiol groups(40). These ageots actiyate taQ
eu,ae br inducin<j change in its confor.ation (14 ••
Studies of intrac.llular distributioD of dibydrofolate
rainctase in rat liyer shows that the anzy.a i5 .ainlJ
prasent in tbe cytopl.asa and iD the aicroso.al frilction ;)f
all e xtracts (14'.
1 O-CBO-B 4PteGlll synthetas.:
10-CBQ-O,PtaGla syotb.tas. catalyses the ATP-depeDdant
foraylation of HliftaGlu at the 1-10 position (29) (Reactioo
J)
10
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HCOOH • RIlPteGIa • &'f P~ 10-CHO 611PteGla • lDP • p~
(Beaction 3)
rhe reaction 18 stronqly inbibited bJ (-, lO-CH 0 Rea Pte.Ha
but Ilot br (+, lO-CHO H4fteGla (17,. 'J'his reactioD vas f.Lrst
:>b~er~ed in pigeon 11yer ez tracts (30' and nov ls knOIiD to
be videl, distribated in tissues and organis.s. In .an lad
other yertebratas. the greatest uounts vere fOllnd in liver
(14). The enzy.e hêlS been eztracted fro. ha.an leucocytes
(111). It bas been shOlln tllat the enzy.e r~uires .onovalent
and d.Lvalent cations for its acti.ity (111'. In the absence
:)f certain catlons, the eDzy.e dissociates into subunits
which are catalyti::ally inactive (61). It bas been rep<>rt~d
that in the rat, the liver enzy.a is iD the cytoplasa (n,.
Tha enzy.e isolated fro. Clostridlaa acidiurici has a lover
K. Ifben H4PteGIll bas 3-g1ota.ates residlles then for
.oDoqlota.ate. The K. value for H4PteGlu 15 O.37X10-~!,
ani for polygluta.ate H4pteGlll 15 0.025110- 3 (68).
5,10- CI:I =84 PtaGla Cyc Iohy drol ase:
This enzy.e catal yzas t he lntercoD~ersion of 1 O-~1:I0
S4PteGl.o and 5,10-CB=B4PteGlu (14) (React.ion 4).
10-CHO BClPteGla • H~5, 10-CH=B"PteGlo • H 20
(leact ion 4)
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..
This reaction a1so lfoceedS non-enzyaaticall,
particularily in tb& presence of cectain ions (14' and foras
botb ,-CHO al&PteGla and lG-CBO UI&PteGla. Tbe reaction Il~s
an aqailibrill. ;,reatly fayollriog 10-CHO H4PteGla (14).
The reaction cataIyzed b, the enzyae i5 siailar ta tbe
Iloo-enz,aatic reaction ezcept that it Iltilizes onlf one:)f
the diasterorisoaars C+, L-.etbenyi H~PteGlu. Tbe I{. of (+)
l.-.etbenyl a4PteGlu for this enzyae i5 7." 110- 5 "and the
rate of tbe back reaction iocreases as the pH La raised fcoa
2 to 8 (1"). The acti Yl.ty of the enzyaa i5 enbancad bJ thl.ols
dn d decreased br P-ch loro.eccuriben zoa te and by lutayy
.etais. Tbe enz,.e bas been deaonstrated in a yariety:)f
tissues incltldin~ li .er, spleen, and hu.an kid.ney leacoc,tes
and bone aarrov.
5, 10-CH 2- H4PteGI u d eh,dr ogenase:
This enzyae cataIyZ&s the ItDP-dependant con.ersioD :)f
5,10-Cd2-HlfPteGlu to 5,10-CH=R"ptaGlu(Reaction 5).
5,10-CH2-H4PteGIIi + 1I1DP~" 10-CH=HQpteGla + 11DPH + H
(Reaction 5)
rb.e IflDPH-linked aethylene 84PteG1u dehydrogenase has baaa
:le.onst rated in extracts of Ehrlich ascites tu.onr cells
(14). The actiYity of the anzy.e is iDcraasad in the
12
presence of
". 211 ()-.
flg+ + ! nd the &a
In llan,
fGr + L-a8thflene 8.PteG~1l 1.5
llwer and kidney cGntaln the
hi~best legels of the dehydr()'lenase. Stodies on rat 11.ger
5 b;>ved t hat t be euz yae ls present in cytoplasa but Dot lD
.itGcbondrla (14'.
t O-CRO HII Pt a Glu syn tbetase, aet beny l Hill> te.au
=ycl:>hydrGlase and aetbylene H4PteGiu debydrogeDase lu~e
been shown (34' to be properties ()f a single polypeptiie
chal.n, ct a III tif ane t iona l protein, as por if ied fro. bo~ lne
ll.~et" (35) sbeep 1iyer (36), yeast (135, and t"abblt
Ilver(136,. Tbe acti~ities of the 10-CHO HIlPteJla
5 y ntbetase and aet b yleDe H4 PteGla de nydr ogenase ha'e bean
detected in e.tracts of coltared hOllan fibroblasts (37)>.
5 -CaO-B4PteGlo cyc10deby drase:
This enzyae catalyzes the couyeraion of 5-CBO IPIPtai;!u
to 5,tO-CH=H4PteGla in the presence of lTP (111,38) (Reaction
6) •
5-CBO aliPteGla • lTP~5,10-C8=H,4PtaGlo • &DP. pi
(BeactioD 6)
5-CRO-OliptaGla cyclodehydrase partial1y
sheep 11yer
1.43110-·8
bad a pB opta ••
for 5-CBO a,pteSlu
13
of Il.8, aDd a
(38.39). &
par ifieci fro.
Ita 9alue ~f
'a 9allle Gf
.114 l
a ".5110- 4 PI lias ob1:aiDed for ITP. At this tiae there 1s [1:)
knolln fun:::tion for 5-CHO H4PteG!u in the cel! eay be becailse
of its high The enzyae is inhibi ted by
P-.arcuribenzoate and bJ '-ethJl.alei.ide (39) and the
inbib4t ion is pr eyent ed by the presence of reduced
-jllltathione (38).
1 "ethylenetetrahydrofolat e redactase:
!etby!enetetraàydrofolate redllctase is the only eluJ.e
tbat catal,zes tbe syntàesis of 5-CH3 BlIPteGlo. the aain
t i3su e and sera. fora of fola te (26). fro. 5. 10-Ca 2-H4pteGlll
( Heae ti on 1)
S.10-'::l2-B4PteGlu + IADPH + H ;;,===' 5-CH3 84PteGlu + IAD~+
(Reaction 1)
The oyerall equilibriua of reaction 1 is far on tbe
side of aethyl tetrahydrofo!at. foraation (41). 80 •• y.r.
asin~ in Yi tro :::onj i tion S "bich pre •• nt IlDP + accuaulatJ.on.
the reaction is run to the left and is used as Ue standard
ëlSS4y for this YiYo. tàe reaction ls
œnidirectional and to tbe rigbt because in the c,topla •• of
the cell the ratio of I&DPH/I1DP+ is greater tbu one (111).
A s.all back.ard r.action coaponeat bas bee reported iD rat
( brain (80,.
14
(
(
The enzyae 15 extranuclear and has a bigher affiaity
for tAe sub stra te wh en 5,10-CH2-HI6Pt8Glu 15 in tjae
polyglutaaate fora (69) • The reactlon
::o.peUtl.ely by S-adenosylaethionine (161).
15 inhiblted
The inhlbitl.on
i5 partiall, reV'arsed by S-adenosylhoaocysteine.
H 2PteGltl and B2PteGltl poIJ9lu ta.ates iahibit piq li YBr
.ethy lane tetrab y~r ofola te redtlctas e (76). The inh ibitlon is
:;oapetiti"e vith respect to 5,10-CB2-BijPteGlu. Tbe Ki
waltles for H2PteGlu and R16PteG1u decr ease vith Cldditl.on:lf
aa=h gltltaaJl rssBae up to six. !ethylene tetrahydrofolate
reducta5e acti"it, is "idel, distributed in .a.aals and !las
been cbaracterizad and pl1rified fro. a Yariety of tissues
including lher (162) and brain (16l ••
5ethyIenetetrahJdrofolate reductase in culture hu.an skin
fibroblasts, a.niot ie floid celis and blood ly.phoblasts ls
predoainantIy ex~rllnoclear and shares aanJ charllcteristics
Ifitb the enzy.e poritied tro. 11.er, inclDding sensitiYitJ
to inhibition by s- adenosyl.ethionine,ql).
tG-CH 0- a4P1:eGllI dellc JcIase:
lO-CHO H4PtaGIu ~.llCycl.s. catalyzes tà. àyârolyaia of
10-CHO a4PteGlII iDto for_ate and B4PteGlu as •• ow. bJ
[eaction 8 (S,).
1 G-CHO a4.uGla + 820 ;,==' BeOO •• 841»t .. 1a
1S
(
(
, (Reaction 8)
Tbe enzy.e is acti yated by catalft ic a.ounts of IlDP+
or IlD., and ls inhibited by p-chloroaercllribenzoate and
eKcess a.ouots of the reaction prodoct, B4PteGlu (54).
lO-CHO n"PteGla dehrdroqenase:
rbis enzyae vhich catalyzes the irre .. ersible (52)
CODye rsioo of lO-CHO tt" PteGl Il to H4ptaGla (52,53) has beea
isolated fro. piq li'fer vith the optiau. pB about 1.8
• (Reaction 9).
lO-CHO Hl&Pt..Glo+ .IDP+ + 820 ~B4PtaGl\l+ C02 + .IOPH + B+ "
(Beaction 9)
rbe react ioo is stronqly iDhibi ted bf both i so.ers of
tt"Pte"lu (52). t'ha 80zf.a is probably identical to 10-;:80
a"PteGIQ deacyclasa IIbich cataly ses reaction ( 8) • but
stoichio.etric a.ooots of .ADP+ are req Ilired (52) • Porayl
!111PteGlo dehfdrogenasa acts as an outl« for ezcess $f
:loe-carboo onit.s ower d.aélJld and is blocked .heo supplr is
liaitiR9 (55)
S-Poraiaioo BIIPt.Gla cfclodeaaiDase:
16
(
..
This enzyme catd1yzes the conversion of foraiaino
34PteGl.u to aetflenyl H4PteGlll (Reaction 10).
5-CIIIIH-B"PteGlu + H+ ~5, 10-CH=H4PteGlu + NH3
(BEact ion 10)
r he enz yae pUl:'i fie d fl:'oa pig 11 ver i s stia ula ted b y K+ JC
NH4+ (14). H4PtaGlu is a coapetitive inhibitor of the eozyae
(29) •
Fol:' aiai noglu t aaate foraiainotl:'a nsfera se:
The deqradat'ion of histidine in aaaaaliao 1i.,er leads
t a the faraa tion of N-forlliaino-L-qlllta. ate. In li.,er the
foraiaino qroup is transferred to H4Pt8Glll (29)>. rbis
reaction is catalysed by foraiainoqlutaaité i
for aiai notransferase (Re acti on 11) (29).
For aiainoqlutaail te + HIl PteGl u~ Gl utaaate + 5-CHBH-H4PteGlu
( Reacti on 1,.
r he enz yae has a br oad pH opt laua and ls inhibi ted br Cd •• ,
80++, and Zn++ (29).
F oraylg .lutaaa V 84PteGlu transforaylase:
17
(
(
r his catdlyses t r d Dsf or Il y l a tl on
')-":dU-H4PteGlu to L-ylutalldte (React1é>n 12) (14).
')-':riO-H4Pter.lu+ L-.j lutallldte ~ H4PteGLu + Ii-farœy lyLutaladte ~
(Reactlon 121
rlle eqLlilibrl.ulI favours the for.atloD of 'J-CHO-A4PteGlu. rhe
c!Dzyœe lS pre'sen t hotll ln the cytJplas. and IIltochondr1d J1
[ cl t 11 ver (14).
The l-ist t lia enzylles, PJ rll1111n 0'llutc1a.1 te
t J r Il 1111 n ot ri Il s ft,! r:l s e d 1\ j f J [ III yi -j lut ,UI dt e t rd n 5 t or Il y l d se t r o.
fllj llver dre on t h~ SdlllE' pept tr1e ( 4')) •
18
React.l.ons involYln~ transfer ot a folate
side chaln ta tbe non-folate .olecule
rhya1.dylate Synt.hetase:
ThlS catalyses tn le lIet.hy13t.l0n
ie~l:yllredylate ta fora tby.ldylate(8eactlou 13). The sourc~
of the aethyl yfOllp 1.S 5, 10-;: El2-1i ij Pte G1 o. l'1g++ 15 rejulred
t or t.Ile bdcterlal but Qat the
J Y fi t Il ~t a s~ (4).
iU~P +,>,10-CH2-HIiPteGte~dT~P + H2pt~(au (Reatlon 131
Thyaidylat.e synthetase i5 present ln aost tl.ssues. ~n
the folate pat.hlldy, thls enzy.e is unique because l.t
";:i t il l Y se s bo th tne transfer ot a one carbon group and two
~rotons to yuüd :lTfIIP. This reaction produces H2PteGlu wh1.:::h
IIUSt be reduca1 by 11.hydrofalate reductase ta HI6Pte:iIu
befora l t ca Q be fUQct1.onally active. T h Y al. d y lat t:t
.iyllthet.dse actl.V' Hf inereases vben eeIIs pro1ifente (7BI.
ln the DNA-synthesis phase of eabryo fibroblast cells
dlllydrofolate reductase, thY.l.dylate sfnthase and four other
ellLy.es associated vith DIil aetabolis., .oye ioto tbe
nnelear regl.OD and in Gl phase they aove back to ttbe
( cytoplas. (33). In this sJste., the diphosphate of TftP 1.5
19
. ~.
1 for a~d f r 0 a i cr I!P •
sulfh,dryl lnhibitors preYent bindlllJ of dO"P t~ lbs
L-=asei eozy.e but not ')~ 10-CH2-H"pteGlu. Tilis suggest. tbat
tllece lar be tvo separdt€ bindiog sites on the enzyae (6~1 •
The L-case.l enzyaa has a pH optiaua of 6.')-8 and ~ KI
v~l u~ of 10- 5 " r or '), 10- L E:ll- BQ Pt aG l Il (2 ~) • fbe
polygluta.ate fora of ')~ lO-C1i2-EfQPteGlu i5 aore effective dS
suustrclte than 15 t he a on oq l a ta aa t e :i ud l t 5 ft ais j t 1. • es
less (&5).
Kinetlc sttldleS conducted on thyaidylate srnt.het.~se
lsol:it~d troa naaao leukeaia blast cells indicate tnat. toe
sY'ntbetase binds dO"P betore bindinq ')~ 1O-CH2-HQPteGla (ObI.
~ethlonine syntbetase:
rhe COnyerSlOn of ,)-CI:Il H4PteGlu to rl"PteG1u 15
::~talysed by the enzyaa aethlonine syotnetase(Reactlon 11.&).
':>-;:HJ H"PteG1u + Hoaocysteine~B"pteGlu + !lethion~ne
(Reaction 1 Q. The iaportant product of this ceaction is aethioIU.oe
.. hieh 15 incorporated into protein and i5 cODyerted ta
S-!deoo5ylaethiooine by .ethiooine adenosyl transferase.
S-adenoSf1.ethionine is belieYed to be the .ost i.portaot
.ethy1 donor in intrace1101ar aetab01is. of .a .. a11an :::e115
( :ind ~5 a precarsor of polya.ines. In y it ro, aethion.1ne
20
1 syntbetase has been shown to require catalytic aaoWlts:lf
(47) and a
c:>bala.10 ("7) vtlich foras aethylcobala.in, the final .etll,1
ionor ln tbe reaetton.
l!ethionine synthetase 1.0 E.coli bas been extensl.yel,
;t.ldled. In this syste. tbe .ethyl group Eroa 5-CU3 BqPte~lu
lS transfer-rad t.:> Co(I)balaain t:l for •• athflcobalaal.n IIlth
:; \1 o5eqLl eDt rapid • ethylation of bo.oc y steine • ls
':0 (r) bala.in oxidl1:es ta Co (lI) bala.in, S-adenosylaetbl.:)(lln
15 re-1ulred ta .ethylate it ta torII .~thylcobala.l.n.
l!atbioOlne synthetase activity i5 progressiYely lnhibited Dy
ln;:\lbatlon ln Dlt[OUS aride whien ir-reversibly l.nactl.Yates
t be Co (1) bella.in bouod ta the enzy.e and blocks aethionine
( synt.hetase.
The aetabolic ::onse~oenc:e of cobala.in deficl.ency
[:)(.5 the basis for tbe 'aetb,l folate tra.p hypotbesis'. III
19b2, Herbert lni Zalusky (129) reported that tb~ ser\l.
la,al3 of 5-CH3 H"PteGlu vere eleYated in patients vltb
:1aflCl.ency of nta.l n B 12 (11). Tbe trap Il as post\Ù.ata.i ta
l. DV 01ye con l'ersion of 5, 10-CO 2- B4 PteGIa to 5-CüJ H"Pt6l.:i 10
.. bicn is irrel'ersible onder physiologl.cal conditions (721.
Ln lef1.cieDcy of cobala.iD, inhibition of 5,10-C82-0ltPte.:ilu
cejuctaSe by S-~denosJl.ethionine aa, also be di.inished as
a resillt of iapairaent of aethiœioe synthetase (12). 511::h
::8115 are deficieDt in total folates, lncluding 5 -cal
( li .. Pte'; lu , p roba b If becaQse 5-CHl U4PteGLu 15 a paor
21
.'
l ~ i
(
(
sUbstrlte for fo13te polyqlotaaate syDtbetase. lccuaol.t~on
of folates vithin the celi appears to reqaire polyglot.aate
S yntbesi s (19).
Ser~n~ hydroI' .atbyltransferase:
Ser~ne hricoxy .etbyltransferase catalyses a react~on
tlut can be considered dS tbe first ~n a .atabolic sequellce
)E steps involva:! in providing aethyl groups for tbfil
Dl.:>syntbes~s of purines, thyaidylate, aethl.onine, :lad
cboll.lle (Reactlon 15) (49). This reaction .akes awailable
the Deta carbon, C (3) ,of serine as 5, 10-C82-0ltPteGlll in tbe
toLite .etabolic patblfaf (48) • Enzyae-bollnd
Pfc~doxal-5'-pbosphate lS a re~\1ired cnfactor for the
reactl.on ("8),
Serlne • H"PteGlll;,===~' GlfCio9 + 5, 10-CH2-B16PteGlu + fl20
(Reactioo 15)
vitb tbe rabbit Uver anzy.e, 5-CHJ 8QPteGllI and 5-:00
H~PteGlll bound witb equaI affioity st the folate bindinq
site IIhen coapared to the sobstrat~ o_PteGla and
5-CBJ o_PteGlo aBy be considerai a
physioloqical inhibitor since its concentration in the =ell
exceeds that of o'PteGla.
lncreasinq the length of the polygluta.ate side cilain
22
:lf 5-CH3 H4PteGla increases the affini tJ of the serine
H"PteGlu (n. by:lrozy .eth,l trilDsferase for S,aJ
dQrlt'dtiyes(7"). It also increases the affinity of the
enz ,.e 5-CH 3 H"PteG lu (n) binary co. ple z f or glycine. Cil y=il1e
1.5 bound 3]-fold aore tightl, to the enzy.e-CH] PteGlu (Il)
co.plex than to the enzy.e-CH3-PteGlu co.plez (1".. It i5
knovn that tbe K. of pol ,glu ta.a te H4P teG luis lover thall i5
tb~t of the aonoqluta.ate for this enzy.e.
Barly stadies in rat Iiyer bad deaonstrated tOdt
soar1ne bydroxy .ethyltransferase
b::>tb cytoplas. dnd .itochondrid
act iyit Y
(50) • 'l'b e
vas present in
ai tocbondrLll
al1Ly.e 1S located Ln the .itochondrial .atriz and 1S si.11ar
1.11 structure ilnd .echanis. to the cytosolic for. (491.
Stud1es of autant Chinese ha.ster oYary(CHot ceIIs vllich iid
not haye .itochondrial serine hydroxr .ethyltransferase,
iodicat e inYolYe.ent of this enzy.e in the glycine cleayag~
syste. (Redction 1&) (26 ••
Glycine. H4PteGlu +11D+, "S,10-C82-H4PteGla+ l'DU
• :02 • IH"
(Reaction 16)
rhe intereonyarsioD of 8'PteGlu and 5,10-CS2-lMptaGlu
in li yer .i toehoDdri a iD .olY89 CJlyeiDe sJDthesis anel glJciae
cleaYilge vith transfer of the alpha carbon of glJel.e to
114Pte:.lo to for. S,10-C82-U4fteGlo(SI). As a.pactael, tàe
23
1
(
(
glycine cleaTaqe sfste. rsquires pyridoal phosphate f:>r
ilo-tl.yitf. Glfcine sfnthesis fro. 5.1o-CH2-HlIPteGlll as
Slib st ra te reqQires C02 and a •• oDia fiz atioD (51).
;lR tcansfor.ylase and IIC1B transfor.,la.se:
Polate deri.,atiTes eootribute C(2) and C(8J in ths de
noyo biosynthesis of thé purine ring by tvo sepaclte
tnnsfor.ylation reaetions (Reactions 17 and 18) (26).
GIB + 10-CHO HlaPteGlu.,==~·PG1B + H4PteGIu
(Beact iOD 11)
AlClS + lO-CHO H4PteGlo;:,=:=' P1IC1B • alaPteGlo
(Beact iOD 18)
The eonyersi on:lf GIB to PGU 15 catalysed br :;IB
t raDsferase (26). This reactioD aediates an irreyersl.ble
tnnsfer of the one-carbon \loits froa 10-CHO 84PteGIu to the
ter.inal aaino grOQP of glfcinaaide riboDUcleQtide. This
for.,lation reqllires for.ate and H4PteGIQ (60,61). vith a 'a
vallle of 5.8110- 5 ft for folate. The eqllilibrlll. for the
ceactioD ls far in the directioa of S4PteGIQ (14j.
5.10-CH=84PteGla vas or1<)10411f idantified as the for.yl
JODor for GiB traD.for.ylas •• It 1a now ItnOlJD tbat 10-:110
H4PteGlu is the trlle for8Jl donor for tIlia reaction(75t •
(
(
ArCAB tran~for.ylase catalyses conversion of ArCAB to
FUCAS (26'. This eoz1.e 15 sti.lllated by K and the pH
o pt i a a. for 1 ts a c ti y 4 t 1 i s 7. 8 •
lO-CHO H4PteGlu dIe equall, dctive as
crade preparations tran5for.ylase. "hen
5,lO-CH=HQPteGlu and
for.fl donors ~ith
tbe parified enZJAe
lS ased in tbe prasence of aaleate bof ter it i5 co.pletel,
:ipeci fic for lo-CHO H4PteGlo (lia).
2S
la a a Jau 4 t aM_
S.,ln. Glycine H'.l!dln. formate
6-CH3 -H4 Pt.Glu •• ----
(
Methionine S.,ln. Thym,dy'.'. Pu,lne
Fi'lure 2. ootline of folate lIIetabolislll in lIa •• ùian
cells.
( 2b
1
(
<-
1. J Fol y Ipol yglota.a te s ynthetase:
Deri Ya ti YBS of folic acid oecor in niltuce
pradoainantly as poly (ga •• a glatuyl) deruatiyes (db),
.. ith aostl, penta or hexaglataaate de riYa ti yes 1.n .a .. &11I1D
::al15 (88,89,. The additi.:>na1 glutaaic acid r4:!sidaes are
attacbed to the initlal one by d ga •• a peptide l1nka~e.
Fo1ylpo1yqlata.ate synthetase catalyses a ~g-lTP-depeDjant
addition of glllta.ate to a variety of redoced f:»1l1ta
substrates (83,8b).
The first report of e nzy lia ti c syntbesis
ptaroylpolyglotaaates ln vitro vas by Griffin and Br:JVD
(82). These vorkees established that tetrabydrodigluta.ate
and te1g1ata.ate IIere foraed frOil If"PteGla in the presence
of ATP, flgel ,~lata.ate, and a (164)2 S04 precipitata of
cbarcoal treated E. coli eJ:tract (82).
!a •• alian folylpolyglata.ate syn the tase ba 5 baeD
parti~lly parified froa Chinase baastar oyary (CHO) cella,
cat 11ger and hog liyer (81). In aIl cases, the enzy.e bas a
pH opt.iaua and is tbe prefarrad
pteroyl.onoqlat.aaate substrate. It appears tbat a single
3ynthetase can catalyze tbe step-Ifise addition of glutaaic
acid residues to redlJCed pteroylglutu ates (84,86).
St adies "bn the folylpolyglutaaate syntbetase of
:oryoebacteriua sbo •• d tàat a reduced pterrin .oiety appears
ta ba assenUal for effectiye biDclin<j to the folate site. In
21
(
~ddition to reduced pteridine, effectiye binding ~lso
re~oires the c;:,rrect configuration of the glllta.ate a01.ety
lnY;:,I,inq a cooperatiye effect resolting fro. 2 lov affiaity
bind.ln<j sites, "' pteridine sitti and a glutuate site (81,.
The iaportance of foly lpolyglo ta_a te foraa tion lias
been cleaonstrated in a cultured Chinese baastar oyar, (CaO)
=811 .utant. Thesa cells lact fo1y1pol,g1uta.ate syntbetase
~cti, l.t Y (8S,. A.lthouqh neither folate transport Dor tbe
iibydrofolate ra:ioctase are defectiYe in .atant cells (d,),
the intracellular folate leyel is re duced due t:> an
lnability of the cell to synthesize fol,lpolyglllta.ate.
l.o~ar ltbaically groving vil d type (IITT2) CHO cells '1rOlf1.ng
ln Pt. eG 111 aet aboliz e fo late t 0 fol ylpolyg lu ta.ate of chal.O
length np to nine vith tbe bexa and heptdgluU.a~te
pred08.lDatinq (81,. In vi1d type CHO cells IIp to 90i of
in tr acelln lac folate i5 as fol ,1 polyg l ota.a te.
II' 01, laooog l uta.a te accounted for oYer 901 of tbe
lntrac~llalar folate l.n autant cells (81,.
roI, lpolyg .luta.a te s do not cross, or are onl, pooe l,
tcansported across .ost cell aeabranes (81,. Coosequelltly,
aetabolis. of folylaonogiutaaate to folylpolygluta.ate foras
f Ilcil il at es lot racellaiar accu.uIa tion of f o.late b, aan J
cells (87' .Polylpol JCJ.l1l ta.ates are as affect he as, and in
.an, cases aore effect he than, pteroylaooogiutaaates as
substrates fOf tbe eozy.es of on8-carbon aetabo.lis ••
C o.parisOD of io tracellll.lar folate concentrations
28
r9~l.li.red ta perait celi Clrovth and la yaloes of aost folate
Bozyaes reyeals that for aost eozy88s, the concentration af
1. nt ra :::811111 a r fOlate vhich sopports qrovt h 1.5 100 tiaes
lover than the Ka for aonoglutaaate folate. Ka's reported
for polyqll1taaat.e fo1ates are si.ihr to reported critLal
l.Dtracelll11ar folate concentratians for qro.th. It vo~ld
tblls appear that poly gll1taad te f ola tes il re the na toral
sl1bstrates for a~st tolate-dependent enzyae reactions, and
that these reactioDS wOl1ld not pcoceed vith;>ut
f o1ylpolyglutaaa ta tola te. The aaaaa1ian enz, _es not
1eaonstrating auch qreater affinity for polygllltaayl than
aonG<Jlut,aayl fo1atas are aet hion iDe syotbetase,
dib,dr;>folate cedl1ctase, aod fol,lpolyqlutalate synthetase ( \ (86) •
Tbe aechani5a by wbich po1'Cllotamtes are better
utilized as $obstrates are Yaried. Tbe polyqlotaaate cluin
L5 often osed 001, for increased bindiDq as eyidenced br a
decreased Ka vith no difference in '.a z (86). 10 tb;>se
reactions vbere either botb Ka and 'a lU are c haoqed or onlf
Vaaz is changed, the polygl~taaate cbain al15t a1so infloence
the aet he site, perbaps by induei ng confor.a tiona1 cbao~e
(66) •
It bas been de .onstra tad Ua t fo1yl pol Jq 1 otaaate
sllb st ra te is channe1ed fro. actiye site to aeti ye si te io at ,
19ast one .ultifunctional enz,ae vithoot release of tbe
( interaediate produets. Tbis pbenoaeAoD .as Dot obser.ad vith
29
aODoqlataaate sabstrate (87).
30
(
(
1. LJ Sa •• alian folat e tta nspor1:
l\icroorganislls and organisas 4Ihich are not able to
syuthesize folate, bave a sys1:e. vhich can taken up folate
fr-::>/I the environllent and transfer it into the cells. Ttlis
systell appears to be ca rr-ier-mediated and lIay
fac.llitated diffusion.
Transport of fo lelte has been studied
function by
in un 1
aUKaryotic and prokary)t~c s1stells. In 110 st .allllalian ceIIs
5-CliJ H4PteGlu and 5-CHO 1l4PteGlu are transported a:>re
effectively then 1S PteGlu (95) .Ho"evel:' in intestinal
ep1thd11ulB, oxidized and reduced .onogiutaaate folates are
transported siailarily (102).
5 olle transport stadies of folate have ossd tlle
4-1Iaioo-l0-lIethyl analogue of PteGlu, .ethotrexate (90),
because t his nteriai vas assu.ed not to be aetabolized
v~tnin caiis (96). Because polyglŒta.ation and aatabolisa of
lIethotrexdte does occur, aod sOlle transport studies lIeasured
ooly radioactivity, soae of vhich lia y have bean:m
lIethotrexa te frag lIents, tran spot' t stu dies a sing tbis:
analogue lost be I:'eevaluated.
Transport of
generally si.ilal:'
(97) • Both types
fola te c:>/lpoonds in Il aallalian caiis i5
t 0 the corrasponding process in bactecia
of ceUs utilize an energy-dependent,
carr-ier-.ediated, active transport /lech~nisa (90, 97~ •
An act iye transpor t procass for the cellular uptaka :lf
31
ta1ate vas [.lrst SU-ll~sterl hy Johns et al. This If as
-> li t.l P-'> rte d by s t u dl e s w1th d mutant _urlne c~ll L517Y wnl.:::h
l S re S l S tan t ta ~ethotrexate (4-IiH2 la-CH.] P t eG lu) ( 1 J ~ ) .
K 1. [l et i c 5 t u die s s u q g e 5 t e d t h d t t.-r cl n s p ü r t Il as_ e d 1 a t e d 0 Y a
carr1er proteln locdlized
ln t;ne lIutant r e s 1 st cl n t c ~ 11 s. S u ch n Cdrr1er bas O~en
lsoldted troll L-CàSe.l ce115, anJ murlnt=' l"uxelilld cell~ 1.1210
( 130)
sh')wn tbdt folat\:! transport l.n lIùrlne
1. 1 .!. 1 a 15 dffectpd by thf>
con:::entrat1on c y:: lu- nUCle:lt1dlds. T heoph, 111ne and
E2 WhlCh enhance the endogenous levei
ut ~AItP jecn~ast!l th\:! rate of aethotrexata transport loto
these cells nptake of
~rythrocytes also 1ncreases after ATP d~platlon (l00), <ioti
lI~taDollc ln h 1 b 1 t 0 r s su ch as aZlde v hleh lover the cll1P
levei eohance .ethotrexate transport. H e pa t oc y tes ( 1 J 11 ,
e['ythrocytes (100) and L1210 (95) bave an act..lve syste. for
e f f lu J: and 1nflux of t clates an d .a t.hot rax at a. .. et a b::>ll.c
l fi h :l b lt 0 r 5 1 i Il e dZ 1 d e 1 n hi 01 t the forlter to a la['1el:" delcee
thdn the latter.
Influx dnd dccu.uIatl.on of .E't.hotreIate in L1210 ::131.1.5
1$ ln t 11lencad _ar kedl y by an 10n s (90) •
repJ:esent.l.nq con::::entratlon f:> r ha l f • aI 1. _ al rat e 0 t 1. n f 111 K
lIere reduced nearly 10-told when Yariolls buffered sall.oe
solut1ons vere replaced vl.th aither REPES or DEPES suer ~ se
32
. < ; ) , ., '} '. ,
'!
( ) 0). 1 t b dS been s ugqest ed t ba t .et b otre xa te tr<l Dsioe a t l JO
lO L1210 .ay utillze aD anion exchaoye lIecbanl5 •. Br ttllS
hY?Jthe5~S ini lUI of folate aoleeules l 5 pa. l Le d li l the f f 1 u x
-lt lntraeellular dnlOOS 5ueb as Pl., lieDJ , and Cl (90)_ 11so
d ~Ilde variety of extrctceliular anlons llile s - C HO H 16 Pte'; l u
:tr.!l AI!P can stlilulate the afflux of folate co.pounrls (:1J).
1 owt:!ver , beea use Ollsothl:Jcyan:J-Stllbene Dlsul tonate a
hl.-jbly speclfie lohibltor of the band 3 cl 010n channel Jf
erythrücyte sll-ihtly redueed uptake ot 'J-CHJ HllpteGlu uy
drythrocytes (10'», lt bas been sU1QesteJ tnat fol~te l5 uot
1 0 L IId 11 Y t L ao s po rte d b Y the a n 1 0 n tr a n sp ) rt s y ste. • Al SOL fi
cOutrist to pbosph'ite transp-lrt, ':>-ct13 H4Pte~lu peraeab.l.llty
d.:Jes nù t ~ppear to correlate vlth lIeabraoe llpid CO.posltl.On
( 1 Ù b)
T he a 0 l 0 n a f f ec t son fol a t e t L ans p or t r es Il l t frua a
j 1. L ec t 1 n ter a ct ion vito tbe aethotrexate transport systea
:j 1. D:: a st 1. • u lat 1. on 0 f t be eft 1111: br 501 occurs at anl.on
.:oncentratlons sl.ildc to thea ICi yallle5 for inhl.b1.tloo 'Of
.ethotrexate ~nflul(99) _ These dat:1 suqgest t.ne sa.e carr-l.CH:
prot\:!in for both etflux and at 101. Tbe fact tllat
p-c.t11oro.ercaripbenyl slllfonate inhl.b~ts 1.n parallel b;)tn
.e tnotJ:e xa te ~nf lUI and efflux further s~pports the single
Cdrr~@[" bypothesLS (99).
:0 .any of tbe st ad l.ed cell l tues 8xcept the
lntestl.oal eplthelia •• the afiiaitf of the tolate traosp:>rt
systea is 100 foU or .ore biqber for reœc .. d tolates than
H
1 for Pte Gl u. It has been suggested that PteGlu and redu.::ed
toLites .ay be traClsp~rted by tvo dlfferent syste.s. rhe
,J!>5 eI:"'at ion that p-chloro.ercuriphenyl s al f ana te inbibi.ts
tL1Dsport ~f .eth~trexat8 f::>late but not PtaGla strengtbeued
th1:; hypothesls. HOllever raeent ohservatlon br Huannelten et
11. (90) sugqest cl 510:}1e carr1er syste. for the rlotduced and
folata. These rasults haye beau obtained by
uS1ng pnrlfied H 4P t eG lu.
(
1.':> Endogenous folate l.n •• uaalian celIs:
Iia.ans ln c;)ntrast ta .an'f plants, bacterl.a, lod
yelst are uDabla ta synthesize folate de 0.:>.0 and depend on
an cld~uate dletary sllp~ly of folate (2 bI. Nor.al foll te
a.Heloolis. aiso requlres pr:>per absorption and dlstributlon
,)t jl.etary follte. Tba source of folate for 1I0St caiis of
t he body 15 the clrculat iog folate of pIas.a, .ost of vlll:-b
15 ln the fora ot 5-CH1 H4PtaGlu (108). The cODcentratl.on ;)f
t.Jlclte in plaslld of nor.al sllb1ect ranges betvaen ') and 1')
DcJ/lll Dut cllnicdl deflclency is Dot obser'led uniess tlllS
valu-=:! re.alns belov ]-" ng/al tor 3-... onthS (101,109).
The folate concentration reqaired for apti.al <jcovtb
15 dlfferent in dlfferent ::e Il li ne 5 in cul.tura.
t;xtracellular folate requlra.ent (PteGIu) for .ul.aai qrolltb
:>f fibcobL1Sts is 5110- 8 fi PteGla which œ preseD t 22 n~/al
(110). The fOlatd requl.re.ent (PteGla) for grovth of • .lusa
leui.e • .l.a cell L1210 (111) and the bu.an leuka.ia cell I{S62
(11l) l.S l ua or 4"0 Dg/al. This deaoDstrates at least 20
tl.aas aore tolate requireaent tor .aIi.al qrollth of tbese
leu~e.la cells ::oapared ta nor.al fibroblasts.
These values reprasent require.eots for eztcacell~lar
PtaGlu as tbe soarce of folate in œltllre aediu., vhicb ls
Q:Jt cl nat ur a l fora of fola te aYa Ua b le to .ost cells in
y 1.0. "ost of tha circulatin9 folat e in plas.a is S-:UJ
( S"PteGlu aod transport of redaced folate 15 100 tiaes • .l[e
35
effectiy@ than for PteGlu. It is tbus probable tbat opti.al
~r')lftb of caiis 111.11 requ1.r@ 100 ti.es less redllced fol~te
tnan PteGlu. For axa.ple, ln bu.ao Ieukaa.la calI 11:5&2 111)-.
ft ?t9\illl lias requ irad for opt.iaal grovth, but a sia!.lar
~roiltb rate lias achiEyed vith 20 ol! 5-CRJ-H4IPteGlu.
l'tost intr~::dllulilr folate 10 culturad aaaaalian catIs
!Id, been reported ta he in the fora of folate polyglutaaate
raD~in'l fro. ,)O-60~ ln tif 0 stadias Ifit b fibroblasts 1.n
.lOgdC it haie grovth ta 10-901 in confluent flbroblasts
(115,11&). :0 hu.an ieWleaia celi K561 <j2~ (112),80C1.De
le..akeala cell L1110 q61 (119) dnd Chinese haaster 09ar,
::e11s ( C 110 ) 9 71 (1 20) of lntracellular folate bas oaeo
reported as polyqlutaaate.
It bas been shown tllat alteration of folate supply
;: ould change the d istr ibut 100 of po lyglllt aaate folate lO
5:>.e cells. 10 rat 1iyer tb@ proportion of total folata in
tbd fora of longer chain lenqtb folypolyg1uta..ate· lias
-4reater in folate defici@nt rats thao in falate suppleaented
cats (121). Also po1T<Jlutaaate cbain length in cult.urad
nu.an fibroblasts lias sborter 10 oells grolln io
folate-deficient aediaa (122) and the folate coeazJ ••
~, 10-::H 2-HIIPt.8G1n is found v!.th longer polyqlata.ate cluin
Ln fo1dte daficient tban repleted aurine cells (123).
rbe effect of chaio lenqth 00 fo1ate .etabolis. is 03t
::laar but tbe aboye obseryations aay indicate a role of
pol,glataa4te chain length in the reCjulatioD of one-carb".
36
a et Clboi is ••
In con fluent bu.a D fibroblasts, 831 of intracellulac
folate vas present as 5-CBJ H16Pt eGlll (125) • 45' of
l.otr-acellular folate in huaan lellkeai.a celi line 1(562 vas as
')-CliJ vit h 321 5-C80-84 P teG lu and
tO-:tiD-O"PteGlu (125). The .ajority of intracelllllar f01ate
ln aOUS9 L1210 ca1Is vas ~dentified as IHPteGIIl, 5-:tiu
H4PteG1u dod 10-CHO H"PteGIIl (126,127). There are a nu.ller
of re?Orts shovin~ :lifferent patterns of folate coeozyaes 1.n
rapl.dIy duiding tissues coapared to cesting tissues. Tbase
lndicate a sh 1.i t iroa 5-C f:l3 8"PteGlu to
'foraylfolate ' as tbe proliferatlon rate increases (lB).
rnese changes aa, reflect tbe qreater require.ent for pu.rioe
( ~nd t.Qyaid,late synUesis 1.0 the aore rapidl, diyichoq
t1351le (128).
(
37
(
(
C ElAPTBB 2
Hiqh Perfor.ance Liqllid Chro.atoqraphy:
chro.atogrëlplly as a separation technique lias started
in 1906 b, PI ich a el Tsvett. Tbis techniqlle iDvol.v9s
separation dlle to the differ9nc9 in tbe eyuilibrlll.
dlstr iblltion of sa .ple co.ponents betveeo two different
phises: one, a Il:>.io-) or .obile phase, and tbe otber a
st~tionarf phase. co.ponants havin9 distributions favourin'l ~
toa stdtionary phase aigrate slo"8[" than do those baYing
1 istr ibut ions fayour log the .obile phase .
.ias cbro.:st:?<Jraphy i5 based on vapour pressure aod ln
lli"ld cbro.atograpby, separation 15 based on solubility.
In ll.quid chro.itograpbJ the .obile phase or the co.posit1.on
.> f t ha .obile phase is of pri.e i.portance in tbe
separation.
ln .an, separation processes. the objective ia to
obtain r9so1ution: tbe ability to separate co.poDent 1 fro.
C08~oDent 2. Besolllt ion is tlsually defiDed as the distance
betveen tie peak centres of t.o peau dlYided br the average
base vidtb of the peaks (Figure ].,.
Y2 - Yl
R=----~-----------
11' 2 (Ii 1 -12)
Differences iD peak .ui .. bat ••• n e1ated co.poGeats
38
f r
(
sa lan. ,a, ; = __ u,
a~~ <1 [urlction of t.'le rt:ldtivt! 1if!t!r-anc~ in their absolutd
~~~tr~uution between the rbases.
r. Il'iS lor.q tlDlt! th.tt reduciD~ tht:r
ctromataql.aphic pc1cr.iu,; Ir.dterials VJuld
sh a r ~t! n i IJ -j or ~lut~ ?t=aks. Very small
f.J cH t ~ cl f: S =- eq u ir p l nlç: t ta aC1U.dVt!
liy the latE 1 9LO' S th is Lecar.-B possivle,
~I • .l ~1.<;: Lc1R1o:= IlPLC was coi~,t:d to -J\:Iscrloe I:'apid st:paratl0n 1.11
t~chni'jue ChdDIJ€-d the col11mn
.... l..l~ lt.Lth COhcorMIIl~t~nt lDcreas,== in iu!aÀ.ytical !ipt:&d.
vary ir size dDd Shdt€, soluhillty
-inù ~l'::irtl":.y for otht:r lDoleClll€s, dü.cI:'l.Dliuation betweer. liuy
OHd ol tht=se rropert ies ma y ser v e as t!le
iJ à.31.;> foI:' 'hffecentidl distributl0n vithin à re pa ra tl.OD
~ 'i S ~d III.
(Fi 'Jure J)
39
1 Ion e Keban qe ehr oaat 09ra pby (IRC) :
The deqree of solute reten tian in IBe is .ai011
lependeot 00 tha strenqtb of e leetrostat ie interaet1.on
betileen solate and col uan. The t oree of a t t rd c t ion "1.11
dep~nd on:
l-t be nu.ber of cha rges on the bio polf.er- tba t a~y
lnteract sl..ultaneosly vith the packing
2-the charge density on the packing
j-tbd ionie strenqtb of tbe aadiu.
~-tbe type of ions in the aediu.
Th cee va n ab les in .Obll ~ pha se vhieh at f aet reteat 1.0n
in lE': include (l)ioDl.e strengtb (2)type of displaeing Lon
or lons (l)pH.
The charqa on both biopolyaers and support is of tan
p~-depeodent. The iaportance of pH in retentioo is shown in
protein separation. Becausa of the aaphot .. ie eharaeter ot
protein there is a pH at "hieb protein does not haye net
c b~ rge. l t this pH (pI) one voald eapect that the retentl.on
Jf the protein in the eola.n sbould be shorter than at sny
otber pB, but it bas baen shovn tbat seyera! mnits aVilI froa
tbe Pi so.e proteins show less l Be retention than voald be
eapeeted froa thair charqe. This i5 beeau5e of the charge
assyaetry in polypeptides whieh causes a heteroq8n3us
distribation of cbarqa at tb.lr surface.
(
.. 0
RaYee sed phase eb coa atog rapb y (RPC)
This tecbnique i5 basad :ln bydrophobl.c interactlon
betileen residues 00 the poly.er and th ose on a coillan.
Hydropnobi:: lotaractlon affects the tertlary structure 'lf
the poly.ers, and ion paicing agents are often required for
the elution of biopolyaers fro. RPC. ln organie solyent 1.S
[ejuiced to break tne b Y d r 0 pb 0 b i.e in te Lac t ion 0 f t Il e
poly.er "loti tha :::olu.o. The nu.ber of aolecules of or':janlc
solvent required foc desorption fro. the coluan 15 dependant
00 the strength of the displaclng agent ln the aohlle phase.
IIhen the solute contains potentially lonizable speCles
tbe aCl.d co.ponent of the soluble phase E' contribute to
( r8tent1.0n bJ influencing the ionic state of -the solute.
::::ontrolling ionization of sucface silanols on tbe suppoct ~c
foraing ioo pairs bet"een cationic solutes and tbe acid.
size exclusion cbeoaatoC) rapb J (5 Be)
Tbis tecbnigae se parlltes aol ecul.s bJ 5ize bas.d 00
different per.eatioo of the colaao packiug. The resolYing
Sbllity of siu exclusion colaan is a function of four
factors:
1) pore 901ua .. of the pacltill9
2) packinq deDsitJ of tbe support
( J) pora diu.ter distribution iD t be pacltioC)
(
(
4) vidth of the cbroaatoqra phy pea ka
Tbe differance betveeD peak .a&i_a in SEC increases
w l.t h por e v 01u_e. Pore 9'01 u.e usuall J decreases vben tbe
.e;;harucal
cdpacity
S y ste_s
strengt b
of SEC is
snch as
colu_ns(lJl) •
of packl.og
lover tban in
ion elchanqe
42
in creases. The loadl.ng
othe r ch r oa atogr ap bic
and reversdi ptuse
C H&PTEB 3
Tbe purpose of present stad,
Preyious atteapts (117,103) t:l stlldy distcibution of
1ntcacelltllar fola tes lIere se pa r- il t ion
of labl.le
t 01 at e an d cell extraction procedtlres vhicb did not
~ cie ~uate11 presa r ye la bi le f:> late and pr-Qvent
loterCODyersioD of folates.
As aentiontti sarllec .ost ::lf the l.DtracelluJ.ar fol:lte
tn fior-oblasts 15 ln the for. of folate polyglutaaate wita d
Y,rl.ety ot chain lengths p:Jss1ble f:>r sach coenzy.e fc)[·a.
"':hroaatograpby and identifl.cation of folate is thus .ery
ll.ffl.cult vithout COD yersion to folate aonoglata_ate.
ca.lled conjugase wbich bas been prerared froa a variety of
sources. Plost of tllese snzyaes are act iye onlT at lov pH and
lost :>f the reaaiuder hydrolase folylpolyglllu.ate to a
sQorter folylpolNltltaaate but not to aODoqltltaaates.
Becuse of instabi lit y of _ost f olates data g_arated
J31.Dg techniques preYiously eaployed prOYide inforaati.on
about = haDges of relati yel y stable fo lat es. ot har folates
laclnding a'PteGlD and 10-CBO H'PteGla coule! Dot be
reproducibly preser.-ed 411rin9 roatin chrœatognph J. fae
( idant1.ficatioD of so .. of t.e foras of folate iD biologieal
'l
(
aateriai has been a pr able. due to inadequate separ/ltl:>n
Ilsinq lon eXchaD'lars sucb as DUE Sephadex. The developllent
~ f h ig b peLf oraa n ce Iiqu id chr o.a togr aph y pro vided a
separation technique vhich peraitted exaa1nation of
iotracellular folate.
The purpose of present stad, has béen to develop a
cbro.atoqraphy technique dnd dn extraction procedure f.:>r
folate vithin livin~ calis vbich couid overcoae the probleas
1. n p r ev l. ou s st u dies •
CHA PTER 4
PlATERI ILS ABD I!ETRODS
Cheaieals:
5-C1:f3 R4PteG1u, 5-=RO H4Pte;;1u and Pt eG lu" e c e
puccbased fro. Slglla Cheœlca1 COllpany, St. Louis, l'to.
DL-H4PteGlu in 0.5 ~ bata lIereaptoethan01 vas a g1ft Ecoli
J c. H. E. l'Iaekenzi~. Departllent of Bioebelll.stry. Il eG 1.11
University. Tetrab~tyl a.moni~. phosphate (PIC & reag91l tl
lias purchasej froll Water's Assoelates, Milford.
~d:;sdehusetts. All dilutions of folates stock solutiontJ lIere
ln HPLC grade vdter contal.nl.ng 0.2 M beta lIereaptoethell:i1
( i fi j 5 to r e d a t - 20 0 C exce pt toc R4 PteG1u Il bien vas Ilsed
fresh and B2PteGl~ IIhich vas kept under vacuuII in 0.2 ~ bata
lIercaptoethanol. Chareoal treated rat serua and nuaan sarua
lias prepared by additl.on of 5.g of Carbon Decolorizing
Helltcal (Fisher Scientific Co.pany, Fair La vn, Nev Jersey)
to 1 III seru. and c8ntrifuged after 20 ainutes.
The potassiua salt of folie acid (PteGlu) labe.l.led
lIith (J8] in the 3, 5, 7, 9 positions and vith specifie
actl.vity of 35-47 Curies/ •• ol vas purchased froa Aaersha.
Corp., Oakvllle, Ontario and purified br BPLC and kept
f["ozan at -200 C.
( 45
1
(
(
A t-' r'd r :i tus :
:i Il dt er ' s syste. wltb d flxed ultrdY10let letector
(ili'\ter'_ci Ass'lc., I\assaebusetts,
sd.ple", werp in)ected loto u, JDS2.
a • an ua l 10 Jec t Of Il l t h
butter-.; Jf tn~ lnitlal r;oodltlJO -UJseo
,>st it
IJSA) •
_ - 1 ij
r .:Ir
:tl[).atograpny. The sa.pIe was eluted wlth .ixtur!? foraQj :>t
S0 lu t l JO -i. od o;tlldflol ln
.JIII -etraout.,laaaODluaphospbate.
ThO::! -lradldot s.,ste. 1escrlbej lD T'iDl~ 11 Illas used at
per .lnute. A Il soluUon:;
, J ( -::: 11 r oaa t og ra p tl Y we r <:! r 11 ter ed t fi r J U -1 n
.w ,j deqassed t 0 [ [lltt:!r
col u. n (37-')0.lcrODS.
r ou t.l n .. l 'f •
20 .1Dutes Delorl"
Wa ter' s A -, SOC: l ci t t:; j
use. A
Ine.) lias
ilU il [ d
l1sad
• "
Tabl~ 11
(solyent -j[ddlEOt tor collJ.n ~lutlon)
con..: e fi t r d t 1. 0 D Jt
Tl Ih= (al r. ) Ifdtt:!r 'J <) , et naL 01 etodIlol (1 )
0. 0 El') 1') iL 25
'). a el) 1 ') d.25
t> ) • 0 75 2') 1 1. 7'>
70 _ 0 75 25 13.75
1 ~ ell .;u l tu r e : 1
J .•
l Nor. a l f i b rob l ci st st r a ~n "CH - 18 was 0 btained fro. t ne
I1dt>J51lory for Llne ~f tbe Itontreal Chlljr~D
i 0-" ~l ta l a n cl usai for less tnan lO <jen~ratloDs. The cldl1s
fr oz e n ln 11\~uid Dltroqen ln O.~ al of aedlua
(IUO:t'I) con ta ln ln'1 10' Glycerol olt a concentratlon .H
1.ù-2.011ù 6 c~~ls per 1.2:al cryula l}LhS vlal. The ctJlls
wl e cel Il p n t h ci Il ed :ind ~rolln at hlgh denslt y ln lIonoldyar ln
SQrtace area (Flow Labrdt:lrlIdS,
loc, ItlsslsSdUqd, Ont.) an d r e f e ct ev E' r y lia y s III l th 20 al
Jt culture aedlua ln an ataosphere of '>1 :02 dnj <.)5' cllC.
~ulture .ailu. WdS Ragles
~lto non-essentldl daino dcids (Gra nd Island
~ oa pa u y, :; r and zr 5 l an i, 'L 1 .) Wn l ch Il as
10' (by volu.e) f e ta l calf se rua (Flow Labrat~[l.es,
l'tl;i.:ilS3duqa, Ontarlo). Th!. S • ed i u. con t.a l ne dL. j Il'' Pte~a u
inj 100 uf'! L-.etblonln~ (table 111) •
folate deficl€nt • ed 111. wa s purc ha sed fro. :;r;llld
LsIln:i Bloloql.Cil Coapanyand "as ldentlcal w1th coaplet.e
_edlU. but dld not con ta1.n foll.C aCld. Pet.:ll çalf serll.
iiied ta the folate 1eflC1.ent .ed1.ua vas Jialyzed
n ou r S l t (J 0 cal al n st a 10 t::> 1 d y::> 1 U ae of O.~' Il ae l III t b tif 0
chaD':les to reao~e 1011 aolecular weigbt: ca.ponents SUCll as
tol;lte. Cultll~e _S11118 vas st.erillzed by tiltc-at.ioD throuqn
( a 0.22 ~ al.llipore fll ter and ali qQots ae re stored foc d
1 1are; it 31 0 C bef:>["1è US~ to datect. conta.lnat~on.
(
49
(
TABLE III
..:oftPOSITro" OP "::>DIFIED EAGLE'S 111111 'UL ESSEIITI1L ftlWIllfi
C o.ponen t lael K Cl • a R 2 PQ4 , H 2 ü
" qSO ", 7 R 20 C,.àCl Fie(NOJ) J,'1H2CJ .; \J.. IlCose L -\A ~ q in 1 n ft L-CJsteln9 4. -Ilist idiDe L-G lataaiDe L- IsolêQcine L-Leucine L-Lfsine L-fIetb1.onine L - Pb en fIaI an 1. n e 1.- Th reonin8 1. -T~f pt opban 1.- T,rosi ne 1.-'a1ine Ch oUne Cl In081tol Il icot ina ai id D-Ca Pantotbenate P, rido~a l tlCl a ibof1aY iD ThiaaiDt! flcl PteG1a la PJruyate P benol II ed
ConceDt~dtioD (.q/.l) 6800 ~OO
1110 200 200 1000 2500 105.0 24. 0 31.0 292.0 52.5 52. " 58.0 14. <}
32.0 168.0 10.0 lb.O ~b.O
1.0 2.0 1.0 1.0 1.0 O. 1 1.0 1.0 1. 1 10.0
(
Extraction of intrdcellll1ar folates:
.0ra.11 fibr:.blasts in confluent grovth lIere incuDdtad
"lta ù. 1 Ill! r Jti }PteGlll and 20 .1 of f:>late free .ed1.u.,
:; Il ) tJ l etI e 0 t ed vit b 1 01 (by volu.e)fetal calf seru. for J
la ys. On ia Y 3 floroblasts lIere lIashad three tl.es "ltb
~b:>spbate buffered saline (Table lV) at roo. teaperatllre and
Leledsed tro. the cultllre vessel by exposurE' to 1.1 ,>1:
ù • 2. '), t r y psi n (Glbco LaboL'-1torles, Grand Island. ILl.) toc
7-10 al.nutes dt 3]0 C. Tbe trypsl.O vas neutrallzed 1I1.th 15
.1 of .ediu. contal.nl.ng fetal calf seru. and an ill1..ju:>t
rttajvej for cell .:ountl.D~. Tbe suspeOSl.OD lias vashed taree
t 1..e5 dt roo. teaperature vith phosphate burfered sallne Dy
.;entrltuyatl.on for 10 alDutes at 300 1 g. Aft er t be ~ast
lIasb the cell pellet VdS resuspended ln 0.05" potas1.u.
phosphate pH b.5 contaiDing 0.2 beLl .ercaptoetbanol. fhe
l.Dtra::allalar folitl:! lias ra1edsed bf OOe of the 1:01101111.0<j
tJ r ocedu r es:
1) r •• erS1.0D of the suspension in a OO1.1lD9 vatee bitn
dod b01.luq for 5 .inutes tollovad bf cooling in iee vater
foc') .ioutes.
2 ) Sooieation (Branson Son 1.C POiler, Oanourr,
:oODect icnt) in 1.ce lIater witb three 15 second bur~ts
separated bf 10 sacoDd int~rYals.
J) Freez log and t,ba.i Dq rap 1.cllr J t iaes in il drr iee
icetoDe aixture and •• ter a.er 8-10 IliDQtes.
51
(
:e11 hoaogenates vere centrifuqed at rooa teaptirature
15 ainutes at 1000 1 9 to reaOYe partic1es and tbe
suparDdtant was locubated with 101 (by yoloae) of charcoal
tceated rat serua (BSCT) at 37° C to C00gect folate
loto aoooqluta.ates. Aftec tlüs
:; on )U'ld se treataeut, protel.u lias preclpltateri Vl.th 70t.
lethallol (by voluae) and proteio ceaoYed by centrlfugatl:lo
it lOOO 1 ~ at ~o = toc 15 aioutes. Plethanol vas evap:lC<lted
l. n t 1.) Il l ng DltrO:J9n gas at 31° C and tbe saaple lias
resuspenrle1 in 2 al ot 0.ù5 ft potassiua phospnate pH 0.5
C ;) Q t a Ln l n q O. }. " aecc apto et benol • Th i s suspension lias
tllti::!ced tbcongb ! 0.~5 Ilccon aeabrane tilter, lias .1Ied
IIHh lO ul of each fo1ate standard (100 uq/a11, and IIdS
lDjected into tbe coluan. The tiae ioterYai betlleeo ::all
ilsruptloD 10d lnjactl..>n lnt.> the coluan Ydcied froa j-j.5
hOllrs.
ln soae eKperiaeots, charcoal treated huaan secua
(US:T) lias used in5tead of RSCT. In tbese eKperiaents saaples
lIece l.ncubated foc 90 aioutes at 310C in 10' (b, voluael .>f
BSCT, dnd protein lias reaoyed by l.~rSiOn in boillng vater
foc 5 al.nutes fo110lltid br centrifu<Jati~ -'----.
To stud, r:ecoyery of intr:acelllliar tolate fr:oa int5:=t
cells, f01ate lias celeased by one of the aboye procedllrQs ,
the b(}aoqenate centrifuged at 1000 1 q and supernatants lIece
reaoyed • Tbe vasbed precipitate lias inœbated Oyerlll.lot
1I1.tb al of b yaaineh,droKide (lU, Boston, sassa., clDd
52
1 ::ountad ln a liquid scintillation couDter in sClntl.llatlon
flllid conta1Dinq TolueDe, POPOP, and PPO.
r.ientificdtl.on of folates eluted froa the coluan:
T 0 a id l nid e nt l f i Cd t 10 n 3 f st cl nd a r:d 0 r: Lad i 0 a :: t 1. • e
[Llctlons of int["~cellulaL folates, eluted fractlons fLa_
coll1ans veLe collected in 0.2" beta aer:captoethanol lod
wec~ ~ssayed al::["~blologlcally vl.tb tvo asself organlsas:
l..à.;tobacl.llus C:!S9l ATCC 7"09 and Ped.lOCOCOlS cerevisla lT'::,-
~Otil ('aex:icao type Culture :..:ollectlon, Bock.,ille, "d.l. The
tOLaer organisa -jrovs on folate aonoqlutudtes, the l~ter
:>nl, on fully-redu::ed, non-aetnylated .onaglutaaates(1l2l.
1.. casei dnd P. eere~isia vere _ ai n tained Ln
_,llnt~nance aedl.ua(133) (Dltco Labratories. I>etrol.t Plien.) •
.ia_ple vere "dded dseptically to sterile toldte free _edlua
alLeady lnnoculated .ith assay organisa (lJij). Sa.ples ware
lncuoated tOL 18 nours at 31° C and turbl.dl.ty was aeasur~d
il bOOna and co.pared vith that of stdndards. Rach 3ucb
~ssay lDcluded standar1 eury.,.s of 20-J60 pg/al of OL-5-CHO
--~----
1
T IBLE l'
Phospheite Buffered Sall.oe (pR=7.~)
N aCl
Kcl 0.2 ':)8/1
K a2po4 0.08')9a /1
Glucose ga/l
Chlorofora 0.1. al/1
!; ",
~ " >'.
2 l
t J '''1l
i .. 1 l' '" , =;
" j: 'k -', t
ta
ft
1 CHl PTEB ')
RESO LTS
Pattern of elutl.on of folate standards f[oa HPLe:
Standard folates cootainlnq P1Bulu, 10-CHO H"Pte,all,
10-.:tlO H2PteGlu, lHPteï.lu, '>-CtiO H~PteGlll, PteGlu and S-':HJ
H4Pte<ilu, prepared iro. dlf tarent sa u rc ~ s (se e c b il pte [ l,
"t! r e ,il l ut e d t 0 1 0 0 u~ III l lO HPLC qrade welter, aod fl.ltered
th[Juqh 0.4'> u aeabr'ioe fl.lters. 20 ul of t;ach standard
toLite lias applled ta caluan sepdrately
j ~ 0 S~ t y 0 f the 8 f flue 0 t a e as ure d a t 2 54 na. Tbt! alxture ~J:
t w J, t bLee, or aor e a f these f 0 la tes lias al.:io
:::broaato<}raphed ta ident if Y tbe sequence of elution of tne
folates. Plg.] lllustr'ites separatlon of 160 ul of a al.xt\lre
of 8 3tandard folates(20 ug of eacb folate •• Incubation :li
H4PteGlu vith a three fold excess of foraaldehyde beflre
~ddlnq It ta tn;! aixture altered tbt! pattern of ellltlon
(dashed 1ine Pl q • J) as conyerted to
'>,10-Cd2-H4PteGlu, lIhl.cb elut&d at 39 ainutes. Based on su=h
:ituùes, fractians elnting fro. the colaaD IIere ldütlfl."d
as follolls:
55
(
3 aiD sol~eDt froDt
& al n a er capt:> et heno l
12 a1.n P1Bqlu
17 .ln 10-CHO H I4Pt eGI Il
20 al n lO-CHO tf 2PteGI a
22.~ al n H" Pt eGI Il
2~ al D ')-CIiO Hl4Pte~lu
314 al n H 2Pteta u
39 111.0 'J, 10-CIi2-H4 PteG lu
"2 al fi PteGlu
,,~ .l. n 'J-CIiJ Hl4 PteGlu
JL lO-CHU HI.t PteGl Il If as qeu 8C at ed
r~IiO EJlIPteGla ln 0.1 N RCI
r or J-I.t bours
1. n cu Dat ion qen era t es ':l,10-CH=HI4PteGlll,
by 1. n cu ba t l n <J Dl.
IIblCb,
1011 pH
Il htill
neutrallzed to pH 7 foc nour qenerdted a u.xture of ~-:I:fO
il.tPte.Hu,lO-CHO HI.tPtttGlu, 10-CHO H2PteGlu. 25 ul :>1 the
~o:>Ye solat iOD at low pH IIdS applled to the coloan. Pl.-lllre
,,_ lllustrates frdct.l.OnS eluted tro. colu.n. Practions at J,
b ilod 29 a1.n utas are solYen t fr ont, aercapt oat hanol !lnd
cesidllal ~-CHO H"PteGlu cespectiyely. "ben tbe ~boY'e
solllt lon lia s .aintalned at pH 7 ror Dour before
elle a.at oqrapby, t he peak at 5 aiD disappeared and nelll peal.s
17 and 20 ai Dutes appeared. Sol .. ent tr~Qt,
aercaptoetbanol, pt.eridl ne, P1BGlu and 5-CHO l:I4PteGlu Il.r. aIs.) oDserY'ed at J, 6, 1" 13 and 29 ainut.es. This indicates
56
(
tbat Deatrali~.ti~n
::',10-CH=H4PteGlu to tllo nev fractions(Pig. 44 dasned 11.nel.
Pollo.log lncubatl.on:li tOt:! sa.e solution at r::>o.
teaperature for 18 hours (Plq. S dashed line th e Ila tee l.;il
dlutln~ dt 11 aln de:::reased, th~ fraction \::!lut loq dt .!O
.inute::i increased, and ci n~v fractl.on eiutlng at 34 alilutes
ci P P aa c e d • The ci b 50 r ~ t l 0 n ::> pe c t ra 0 f
l.njl.cated that tllase prJbably lIere
f r:1 ct l on s 17 d fi j lO
lO-CHO H4P t.eGlu dnd
10-CHO H2PteGlu. Fcactl.on 31l coeluted Vlth H2PteGlll. TlllS
tentatlve identificatlon of fractlons lias confica~d by
ai.:r.)o.l.oloqlcal d3say of the fractions collected ln tUOE:!S
contallllnq 0.2 BI'tE (fl.q. 6). FractIons 17, 20,29 .101 J'4
ciupported yrovth:Jt L. Cdsel, and fractl.ons 17 and 29 diso
31.1pported qrovth of P. cereyiria but other fractlons dlj 113t
::iUppoct ba:::terl.ll ~rolltb. This
elJtloq at tubes 17 dnd 29 as
study l.dent lfies fractl.::>ns
fully reduced folate, .::>~t
probdbly 10-CHO H4PteGlu and :,-CHO B4pteGlu, and fractl:lQS
eiutlny at tubes 20 and 34 as 10-CHO H2PteGiu and HlPteGiil.
It diso confiras the lDstability of 11)-CHO H~PteGlu despite
tbt:! presence of aercaptoethanol, and suggests t llat durl.og
proloDged incub:itlon soae H2pteGlll .ay be for.ed fro. taese
coapoWlds. The P1BGla fractl.on (t ube 13) ~ncreased dUCl.ll-J
lncubatioD for 18 bours iDdlcatl.nq s::>ae instability ot tbe
';i-10 Do nd in one of tbe pcod \lcts. pr obably lO-CHO H~?te(au
but H2PteGlo could Dot be eJ:clllded basad on tbese data.
57
St~bilit, of folate standards dl1cl.Dq call e.ltract.lon:
To stady l.ntra eell ala r folates,
:811 ~.Itracts by treat.ent vl.tb j~eonjuqase enzymes. rosse
enzy.es have baen prepared fro. d1.fferent sources lnclud.ln~
k 1. d n e yan d h U Il a n se ru •• /'tos t of
lct.lVe only at 1:>11 pH. Poiates are lablle and are stabl.ll.Led
routl.nely br ddd1ng red ue lng dgents such as beta
.erc~ptoethanol. Heat ~nd l::>v tJH .ay alter tbe disteibutl.on
~f folates 1.n cell extracts. To deter.lne the effect )n
1l.fferent folatas of ,il.fferent cell extraction and folate
decon )U gat ion proe edures, tvo sets of folate standards
( 100ug/al) of H~PteGlu, ')-CH3 Hl4pteGlu, ')-C HO R4 PteG lu,
PteGlll and a aixture of 10-CHO IillPteGlu and lQ-CHO H2PteGlu,
vere te eat ed as for cell extraction. One set vas incuoated
vlth RSCT ~t pH 6.5 (31), and treated vitb .etbanol to reaove
pr.:>tein IIhile the otber IIdS incllbated vith HSCT at pH 5 :lod
protein vas preeipitated If ith heat. Follov iog
:llrOilatognpby, tba optical density of the folates in each
:3 et lias co.pared. Co.parison of the reeovery of tolate
fra::tions is plotted (f'.lg. 7). cireled DIl Il bers represent
folates added to the .ixture, naked Daabers represent
folates eluted vhieb vere not 1.0 the original solution.
It i5 appa['ent that reeoye['y of 5-CHO H4PteGIa, 5-::83
( H4PteGIa and PteGIa vas si.iIar after both eKtracti~D
58
pr~cedures. PlBGlu lia 5 'Jenerated by both ex tr act l.on
~ r ~ ce dur es in di c a tin CJ destruction of 50ae f 01a tes by
bre~k1ng of the 9-10 bond betlleen pteridine and PABG1u, IIl.th
lIore PlBGlu qenerated by 1011 pH incubatl.on and precipitat10n
of protel.n br heat than by in cu bat l.0 n at 6.5 and
preclpitation of protein with methanol.
Incubat10n a t pH 6.5 wl.th HSCT f0110lled by
prdC1pl.tation of prote1n vith lIethanol perlllitted greater-
["ecove["y of added H4PteGlu dnd lO-CHO H4PteGlu than did the
procedure usinq lncub~tl.on w1th HSCT at pH S followej by
P[d~l.pl.tatlon of the protein vl.th heat. The forller aiso
yenerated two slIall uD1dentl.fied fractions. The latter
proc~jure gener3te1 lIore PABGlu than did the Eorller, and
( 1:>["8 lO-CHO B2PteGlu which is an oxidation product of lO-CBO
II 4 Pte" lu.
The extra:;tlon procedure ut ili 21.0'1 incubation Il l.th
~ S CT dt pH 6. 5 f 0 11 ove d by p~ecipitation of protein with
Il eth a fi 0 l t h usa pp e a r ed top r e se r vell 0 r e ad de d fol a t eth a n
did tbe other procedure.
Stabl.lity of t olate standards duri Dg decon jugclti:>n
proced u re:
(ieneLation of PABGlu in rot h pro cedures in the
previous expeLiaent indicates so.e instability of 9-10 b~nd
( of folates durio:j conjugase treataent vith rat serua and
59
,..>j"
i j
" J l 1
p r ot ~ l Il by 50 ure" 0 i
Dy t rel! t l n ~ ct l f te r e Dt l D dl "1 j 1.1 d .1.
t .:l.l. 1 t ~s t' y
"J5t f~lates tested sur"l"eJ fb ... proc€>duc€> (tablt? V) O"l t c;
p ropù r t l .:lu ) f ) - _" el ,
), lJ-~-I2-H4Pte •• l. J 1 ' .J ~.;lj
~ ; j l t l ,J [ 1 ::; ad 1 1 ~ r 0 p ) r t l J ù
J r H .. P t ~(; i u -, 1 0 - ~ !iL - :-. ? '" ,; l J
) • cl • "'." , {) ut
1 II : t ..., Dl !.>d k .; 0111 r P A ~G i
.1. 1 ~ Ij t l r lei ~b r J • a • J ~ r a a a,
, J l t J ..:: J r. c~ [. t r ~ t l J L .lr l q l LI â l :>lutlOf ~ou .. d
_ dl. <.-Il l ci t <=> 1 _ rl .:. P t~'; lu, IId
o r ~ d k ,10. D P r oà oc t J t 1 J - Cf" G H4 p. to'G lU.
.JI. r !,fCO"<
- ... t J l 1. 1 fi -1 :. JC
, 1 , ")
~) fi r~t" Z l [J -.j d n d • b <1 Il l li ~ r dp l j l Y t lU @Of::' t l a'- "
'" 1 _ ~ • JI, @ Ill .... rt 1:-'" d .. 1 Il a t .. r ) .. ~r ,,-, J • l .ù t t,!
, ,. ~L"1 - , ., t J ~
, 1 y, Il.' r ,) - 1 • ~u r l t " ... j l t'; ? t eJ l Q, Vd; D~:1, : - l ... <i s.? j t ~ j.
r-" J) ~ n .. p ~ • 1. e!'" ,., t ::) J8
~ l • i U:Jt <;;.
ù J a O~'" n i'I ~ ~ Il a 00 • lit:! W <I.:i'htd
t n ... r '\ j l Od ; t l .. l t ,. t tld t lL t'u.
0, ." ~L a
d b~ll.l..n-l " <st er, -;OLl t lcat 10"
~ LI 1 t: ~@z l D '1 d D à • 0 4. Hl q •
eltitct tll>;
on t ilE j l S t.r lb ut l OD of @ndoqf!'ftoQS fO.lAt.IitS.
; .1 ~ t U r8 5 t l br 1.) b la <; t. s ln tlssue culture
" .; J 0 t d lO er Il ec E' .. pur lt led iH) Pt.a .. lu
1 Il.l ,0 al ot t:ü,,·e tr~
id' s. llacYest.., d dD d
~~trdct ed b, OD@ ::lt the ~coceduces cit@d ciOOye. Th@ eItc:,s;:ts
61
'J' (oy
lise;- pH b _ 5 hours il 04111 proteln
tl r 1:1':1. pl t3 ted III t 0 .01 tn a n01. Tb@
, Jb J ~J: traC t lias CO. pa r~ 1 ( Tanb'!
) J: 1. j '! t l OD J r tnclujtnq lJ-_du
• n d' J ru .1 Wl t l t l e1 tI..,ct ton", an d ... 1
• l J J t dt:' ~ III t? r '" 10'': l U \l E'::j ~" 1 l ,1- L A f' • O.Li te.'o' (T "L l ~ W- l )
Jl!tert:'u':~<:;
• J i .J ~ ell • f.> r . ~c:> .P [ y J!
t! J: t [ '1 ct l JI III l' rI
Jt:l~[ prJc8dur'-!5, dL d JI
:, J ~ U d III l l; q .
1.., confira tbat tIl,," 10-201 of :',,10-('8": -riO Pt.eGl u 1.D
rJz,~" ilnd tlllllled ttJ:tr'!cts 1114S pr:>babl, tnlS tolate. SUC&! dL
standards of 5,10-CB2-R4PtPGlu alJttHl
H&6PteG.I. a, dnd Pt e.,lo la.edl.tlt.:!l,
) • 1 () - : R 2- Il IIP t 8(; l u ( "0 • l..D J IIdS [pcbroaatograpb@d 1I1.ttliD JO
(f lq _ Il •. ot t be stand:l!. d
),l()-':i2-HfaPt.eGlu and r adl..:>a ctl y lt .,
1 ~ K t Cd:: t a ppea rai l. n t tl e ln tbe
t r- .H .. '"t lO ns, ldent l. t ied '). 1 ()- C il 2- H IlP t eG l II • PAB.ill1,
,.)t,Hljl.ne (10 alhut.t!sl and au unUlo"n tract1.an @lutlog 4t 2.1
.lniJt~s. .. d b~ l l t Y cl n d of products
; .1l-POCt 5 t hl" ldent l. t lCdt l.on af frdctl':>DS lS
) , 1 J- '- ri 2 - H 4 P t ~r.l u.
r ht:' S 0U rcp J t • b € .i j d 1. t 100 ci • '), 1 (}- H 2 - H " ~t el.i l. u .. 5
111lI...JW!l. l; :.. apr:>bdhl~ t il at l t "dS pc@s.,,-n t l. r . t ilS
. el.. dit r: à ct , ca fUI;Jt be
OP PAH(,lu
1 l. j L i t [ i:o ~ Z l fi,j ~ fi 1 'b '\ ,,1. fi ,j l5 lap['ooaol@
t not J, 10-':A2-H4Pt E'G1.u III <L5 r'resent 1.1.1 tlle DOlled ~I'tract iDCl
·,rJi.l!!o 10llD. A1SJ stady of the stab1.11.tr Jt tolatQ standaC<1!:.
procedure leaonstrate:i tbat a.He
.>r~cedurt'. '-J, 10-CH2-1t4~t IilGle could naYe ar'l.sen t'y ~ llnkn:lui
aeClW.Dl.Sa tr::>a 10-~HC H'4Pt.ebla "hlcb "4S pceseot lO plI:es:o 1.0
131 ... r~sery~d 1.0 baye 'HUtQ
:iur l nq tU t r a ct laD Il lot. D
l t .1..5 pr JOd tie t ha t t nt' '10 -C H 0 -f .:> l cl t p s • or tbe
-;:>Ol':::itlon "e['@ b,1rol,s~d and rec::>.eced 1L tbe c.broaato-.ra •
.:l" t'A 8 .. 1 u •
03
Etr~ct of redac4nq d'lents on foate dlst.rlbutlon:
To dcaaloe t Ile P 055 l bl. 11 t, t h dt l D the t ro z e 0 ~ n d
t III Ile d e l[ t r il c t 50a9 doz,aat1cal1y-a-*dlat.ed loterCOnYerSlOn
Jt toLite .iqht hdY€ occarred and 51.CCe the reactlon b~t"8eD
),1->- -':1 2-HQ PteG.l.. u and l()-CI:W 'i4PteGlu lS 111 DPH/IUDP
j ep en ci en t, th.., e t f ec t J f toese "qent.s lias lnYe5t.11d~1
tlbrobL~st ce 11
l ;, :: u b cl t e d t:J r
PlCH-18,
Vlt Il 10- 1 ft
1. Cl C on fI" ê (1 t
pur l t 1.t!d , j H j
t te • .I. J , tla r y ~1> t ~ r1 , wdshed, ~nd lntrdcelluldr [.:lldtes
!Il OP ,.n 1 1 OP H Il as
1 j::l~a t () t he PIt r -\ct DPt crp
t r ~a tae nt cl nJ p rec l. pl t dt l :>D
.Jr::>t.~lU Illtb aytouIC)l. ':Dtt dll:t.ract vas apphed ta th", caluac
ddded standard tolates. &esults cire shawo lO II1ttlJ.1t ln.,
TH> ... y VIL : t 15 appcirect that tne lo1ate 4::l1uted troa tàe
WdS dltered 0' the preseoce of tllese
:;)D~-=otratlons ~t Dllcleot1.d& 10 to", eJ[t.rilct, .lth 50a., ot
Vl lÀ:> ut tlll:f trdctloDh elutlnq le8s 11.scret.el, than
llu:l.otl:1e. Tbe prtlsence ::>t e1th-u- nucleotlde generated .oce
) -\...Ii j 84Pt &Glu t bdD .ltboat eltber IADP ::u:
IIli>PH. 40 d less '> -CHu lM Pt IotGI u t ban "as ohsened lD tila
these •• lDPS decr9ased :>,10-CH2-liliPt~Glu 10 ttl9
~ 1 t La:: t 5 , ce p 14:: l n q tb 1 S Il l t b d po:> r l , da t UI ti d f r Il ct l. 0 n If 1. t. n
:1 aobll1t., lntecaed1at e b et. " ee D U 2 P t.e Gl q S Q d
:). 1()-:!t 2-H "Pt.eGl q • Tb e a. c.b an 1s. tbe5e cbanqes 1.5
lloclaac, bot because of the effect of nuclaoti des
it If as pcesa.ed that
eOLJae-aediated cbaDges in folates .iqht be occurrl.nq ln
eltracts 'lad tn'lt tllis vOllld be e][~ected Dot to occur ln
el t r d ct s pro cl u ce d by beat "bl.ch preel plta teq
t n l S [ttaSOD, cell eatractl'>D by beat If as
pr-otal.Os. F:>r
:>ttlect.ed 15
;Jotdotl.ally the
.::@11s.
.ost r-eproduclble aaans of dl.sruptl.DIJ the
flle toldte of 0::>ra,,1 t lbroblasts:
To as@ tlle HPLC tltChDlqu~ <lod tbe aDore eatrdctloD 11ld
lè'::..>[, )uqatlon pr:>cedure for studylCJq distrlbutl'>D of folattt
• J[J..HJ 1.1 t aadt e, nor.al flbrobldst strdln Pl:ti-Hl, conf l uen t
lOCUD'lted for j clays vlto 10- 1 " lJH) PteiHu,
Cd1lià3ttd fro. tlssll@ culture
",a,bed "'!th PBS, tbe .e.braDe
~ oOl.l1.ng vater hato and the
yessei "1. th 0.2')1 tCypSl.D,
lias clisrllpt ad by 1 •• er-siao lD
o;][trdct vas i.Jlcubated vlth 1J'
ri S CT ~ t 31 oC f or 2 Il ou [ s • Prot.ein Wél.s preC1.pitat.ed vith 701
(by Yoluae) .etbanol, ceotrlfoged ilnd the supernatcUlt lUS
.irlitd 10 01tr0980 ~as. Tbe resl.due .as resuspendt!d in O.1l5 !'I
pft.>spbate buffer (pH &.5) cODt.~lning a.l " oeta
.ercd ptoetoan 01. F olate stan dards If are vit.h the
d][tract, ~nd lt vas ::bro.atoqx:aphed. The dist.ribution .:>{
L JHj-labelec! tractl.ODS 1.11 silt e][periaents ~s list.ed in Tlble
fHI. The folate distributioD Ifas coDsistent, Vl.tb h.ttla
65
1 'arl.iltloo betv@en st.odies. 5411 of intracellular folate lUS
10 tbe for8 of 5-CHJ R"PtaGIa, 291 as '10-CRO-tolate', aod
le<lsurdble quaot.itles of othee lablle folates .. ere foao:1.
ln ordsr t~ cooti~ ths ldeotltJ of labile peaks
fol~ta 1.0 tva sach e.tracts .. Illch d1.1 Dot lDCLlldtt
,;taodard falate, 81 f ra ct 1. () n S IH! re coll sc te d p e r t ub ~ l. D
0.2" bata Ij,jr:::'iptoattlanol dnd 200 ul of t h ese f r a ct 1. ;) "s
w el e d55 ilT ed • 1. C rob 1. 0 l a q 1. Cd Il Y vit h L-casel and 'JO ul ;)1
[[detian <l 150 C ou n t ad fùr l jH]. The result has u~an
~ Il \.Jill [j l n P 1 Il. q. .J
"
(
66
1
"'li;'
T1BL8 V
ltecoyer-y of ':i.elected foldtes dfter tre3t8en t tor
18COO )U'l~tl.:JD
~ e co. f> q' (j,) P1BGlu (l) Jthp[ j ('1
i:t LPte'; 1 u 10ù J )
'>-~r1U H" PteG1 u 10J
'> -c.:liJ ""PtelÔlu 'J'i 'l ,)
-iL4Pta.;lu <:jQ t, L PflPteGlll)
2 ( '>, 10-CH2- dit
?t~'; lu)
1 J -~ ihJ- H" PteG l u 7b 1,1 12 (lù-CHO-Hlf
Pt eù 111 t
}. (u 0 l den t l.f 1. .,d 1
'>,10-CH2-tt4PteGlll 9') ()
67
- -
Cell Dhrvptton PA861u 10 CHO HItPte61u SCt«) H"P teG lu H/t~lu 5,10 CHz 5CH Unknown' Method Fohtes HItPte61u H .. pte61u
Heat 15 20 5 4 3 2 51
He.t 13 Z6 5 l Z 1.5 47 0.5
Heat 12 27 6.5 J 2.5 3 45
rreeze Thh 11 5.5 4 4 3 ZZ 50 1
r Freeze Th .. 13 15 6 2 2 11 Sl n:
Sonication 18 11 6 1.5 1.5 l 57 1
Sonic.t1on 25 14 7 2 3 46 1
\
TAlLE VI E" ..... ' the _thod of cell dhrupt10n on the distribution (1) of
l 'H J in 'JCtracts of '1 brob 111 tJ lro.n in l'H J Pte6l u.
- -
e.ll Dhl"Uption lOCHO- H .. Ptt61u 5CHO H2PteGlu 5,10-CH 2 5-CH untnawn Method fol etes H .. Pte61u H .. PteG1u H4Pt:Sl u
F reeze- thaw 7 2 4.5 3.5 25 57
F reeze- thaw 17 7 3 3 13 57
F reeze- thaw 14 4 4 4 66 7 +NADPH
(?>
\D
F .... ze-th., 12 4 3.5 14 65 1 + NAPP
TABLE V1l E'fKt an the dhtribution l 'lof'" fra. fibrobllsts VnMl in
llHJ PteGlu of incubation 0' ' .... ze-thtwed hc.agenltes w1th NADPH .. d
NADH.
_urse nr sr If • t* mttmu sm 3 5 'p' .. '~ • • In1i1"~-'" -~ .... ~, " 1 ... , ~ --.ll~
-- •
Experiments , O-CHO Fol a tes H4 PteGl u 5-CHO H2PteGlu 5.1 O-CH;/- 5 CH 3H .. P teG 1 u uni dent f f1ed H .. PteGl u H .. PteGlu
33 6 4.5 1.5 53
2 28 B 2 3 1.5 54 3
3 30 3 2 4.5 3 55 2
4 24 5 7 3.5 2.5 58 -..l 0
5.5 3 2.5 1.5 52 3 5 32
6 30 7.5 3~ 3 3.5 51 2
Mean 29 6 3 3.3 2.2 54
S.O. 3 1.6 ... 0.9 0.7 3 (..
TABLE VIII Distribution of l3HJ-folates fn (Il repeated cultures of nOMll1 human
f1broblasts extracted and deconJugated as re<:OIIIDef1ded.
~-~ .. _--- - ... 4,.'-',.,.. .. ...uc~ •• ~"'. ,~
(
o' o
005
o 025
o
, · , · . · . • • • 1 • • . · . • • • • • • · :
•
2
, · , , , · , · , · , , ,
.. 1 1
, 1
1 / v.:"~ ....,., ..... _.,.
10
3
, '. " I, 1 • . , , '
. ' l ' ,
4
6
'V
20
B , ~
" ':
, , , , 1
9 , , 1
1 : ..
l)
. . . • 1 t l'
1 1 ." •
t ::! :
11
: l ,.' 1
, :: i: : t,'II. 1
, 1. Il 'u' ~' ~: ',.J' , '
30 40
, ~-
50 E Jution lime. (min)
(runt, Il e 1" c a t- t :Je th a n 0 l , J) P'\ BI.i l. Il 1
'i 4 ::> ~ ~r, lu, ~) l v -C Il 0 d2?+eGlu, b) N .. PteGlu, 7)5-CtiO
IJ ) H 2 t' t ~ Glu, 'l) 5, 1 0 -c ri 2 - H 14 Pt ~ Gl Il , 10) Pt a"au,
11) rC!l3 fi4Pt eGlu, • The ldshed l in~ r~ ~re sen t 1"esul te of
of ,1,,+ P t~-;l u fo~d tU cese; or
71
1
(
E c -.1 or> ,.,.
d d
0.05
0.025
10
Lf 1~~1 lnCUDa~lOu Jt S-C:I<...
, 1
• II ~
" " " " " " " " " " " , ' , ' , : ' , '
1 , , l , , . , .
'- "
~ l'
" " ': , . , , , , l , ,
20
Elut Ion
30
Time (min) ,
by ueiJtrallZatioCJ to fil J (dasbed line).
Î2
40 50
., Ï i ,
"]
1
(
o o
0·05 , , ' , ' " " , , " , , , , , , , , , , , , , • , , · , · , ~ , ,
" o 025 " '. , 1
• . . 1 . 1 ,
1 . ' , .
o 10
:' " "
., "
.. "
, , "
, , , , , , , • , , , , , , , , , , ,
20 30
, ,', " , , . , , , , , , , , ,
'.
40 50 Elulion Time, (min)
.) t mlx~ure of lO-CHO
by aCld incutatlon of
J-Ld0 d4PteGlu followir.q oeutralizaticn te pH 7. The solid
111."" r<::f.resents th!:! optical density (254nm) of the mixture 1
Llour a ftt r ne Il t rd l i za t 10n to pr! 7 the dashed
repre~érts t~e chromatogram after incubation of the neutral
IIdÀ.tur~ d+: room t~mpt;raturl::! Lor 13 Lours ir. mercaptoethanol.
ï3
" " l
,
(
o o
E c
n os
002 S
1 :
-:- -- -
o 10 20
. ; , , ,1
. ~ 1
1
_--' ___ ~ _--'-___ "----__ ---'--__ --L-
30 40 so
100(;
E
soo
r J..' J 1 [ :. ' ;. C ~ ).' l ~ L ) ~. . 1 l .1::; .-:;d J ., .... 1-1 :.cur-lrLu .... .1tLL:
) • 2 l r,
ri l t hL. Cci Sf' l (-. - • - • -) , d n j t! •
~ ~ ~ ev l', l l ' ( -- - - -) •
:f'
*'~ }it-
-~
o o
(i OZS
o
7
~.H 1)1 L1SO!.
"'1 tn
(OurlZJIlL.11 i'<1.,), dca rdt
----"--
0025 0·05 00
2,:>L nm
of t l, e 1 J.J..J.t '" [,'cove rej arter
St: r '1 ni cuarcoal
s e r U ID C b d r C Od l t r e il t e d (ver t. 1. cal
tola~e added to tue
mlxture; f\ar..·,~ [I\lmr)\:!r~ rt.!rr8sent Lew faldte gen~rated by the
1) PAlJGlu, 2) lO-CHO H4Î'te~lu, 3) lO-CHO H2Pte';lu,
15
..
1
'J (' " r soo
E 1 !
c
1 -J E .,... ,....,.
I[ ""-
0 :i 0 11-
U
o OZ s .' " Z~ ,1
" , , , , ,1
1 , , . 1
o 10 20 30 40 sa Elutlon Time,(min)
o~ til~ S, 10-C!-l2-H4t't~\u.u
; ., [ 0 rt è t 0 ':J r ct 1 h Y c... t j F ~ L r ù.oJ Id ste e 11 E' L tr :l .;; t 1 CJn~a~;Il~'l fola+..., lac~lleJ w~ttl [JH]. Tue sol~d l~fJe plot.s
(2~4nll) dIlU represeats th\::: stanlard
S / lù-C:12-rl4f,t'?Glll ciddeJ ta t l'e cell extract; tbe dilsned Ilne
folat.e
[ro ... tllE: ce11 f-xtract.
(
76
,
3 1 , 1
1
f ~ 1 ~ ,
1 l(
E 1 1
i "- 2 1 1 !;.
Of , 1 '1 c: ~
'1 QI ; 1
ru j 0
IL. J
0 > .. 1
__ 1 1 ----.J
j 0 10 20 30 40 SO Elutlon Tlme, (min)
I)t t f,(; llstr lLùtlon of [ lfi J-laD~l
j Il l .Il l CI- 0 l.; i 010 :11 C cll1 y I!leasur~d f C ld te ut i 11 2E: 1 L.
cas~J.. (----) ;:roll1 normal fibx:-oblasts lncubateri for 3 clays ln
l LI J lt € Glu.
ï 1
'-- HAPT ER b
Jl::.CUSSlon:
t 1 !J r él b l as t" 1 li cul t U L e
lliueLlted iet~ct:,:Jt lotrace11uLu aetabo1.l.silI. AUDJrJadlltles
:1 d V ..,. u f> f> Il r p pO rte d the Jlstr.lLutlon 'Jt HI t r a ce 11111 lI.
• t:' t cl!J J 1 1',. ( 2 b, 1 1 7) • The ci b no r ad l 1 t i e s ad y d r l se d ue tJ ci
'htt:!::ts VlllCh .ay affect to1ate cont~ùt,
H1Jdt 10n-[ educt lon redctlons, transfer Jf one-car bon uu lts
~uj tolypulyglutaœdte synthpsls.
CJnSlst~nt recov~ry
)t l.utrilcelluldr folate coenzyae foras per.lts exa.lnatl.JO
Jt th~ eftects ot q r:) li th, t:J 1 a tes u f f l C Le n cy, and e f f e ct
.) th ec nut [lents and antl.etdbo1.l.tes 00 lntrace11uldC
t'reVlous atte.pts(117, 103) to stad y the dlstributlJO
lntracelluLlr to1ltes vere coapro • .l.sed by separation
tdchUllues did not permlt the recovary of lablle folates and
cel1 extràction procedures wh.l.ch dld Dot adequately preserve
l.:iblla folate ann prevent loterconverslon of folates.
As lIIentloned earlier most of the lntcacellulac folate
ln tlbcoblasts i5 in the fora of folate polyglutd.dte wLth a
V'H.l.ety of chalO lengths p:Jssible for each caenzy.e forll.
Chroaat ogcaphy and identification of folates 15 thus very
( dltfLcult vlthaut COD" ersi on ta folate aono<jlutaaates.
78
,1 re • ove d b Y :l n t! Il zr. e
nas beeu pr epared fr u. -1 V il r lt.tt '1 of
~.),Hce:;. "ost of these en~y.es are clCtlVP only dt 10" pH !nd
hy1r~lfse folylpolyylu~a.ate ta d
.>QJrter tolylpoly~lutd.dtes but not to .onoqluta.ates.
: n C II b ~ t lC)(l -\ t l 0 Il pH C cl II 5 es lot e r co Dye r S Ion J f 't or_ y l
t .. tr:lllydrJ Eor.s' JE t31ate, "bieb .ay cause accUllulatlon Jt
'>-~d{) ri"PteGlu ln extrdcts of celis. ,)-CHO HI4Pteuiu lS [lot :i
f ùr :i CI Y koC/vn .~tdbollC reactlon, bu t 15
ltlllzed effectuely Dy h II .a n ce ils, and redcts "ltb
decause of l.nstabi11ty of .ost folates data geoer:lted
\lSlU-j technlques pre'Yl:lusly ellployed provl.de datrl ooly about
~n:inyes of relatlvely stclble folates. other folates locllIled
il "P te .; l u :i n d 1 0 - C HO H ~ ?t aGi u co u id Dot bt:l l aliroducl.bly
preserved durloc] routlne cbro_atography. The l.dentlflcatl:>n
of the f ::)[ 115 ~f t:>late in blologlcal material nas
beea d probl e. due toi nad e q ua t e se p il r il t ion us 1. n g l:>D
e lC na n'1 ers suc h 1S DEAE Sepbadex. of b.l.gb.
perforllance liqUld chr o.atograpby provided d separclti:>D
perllltted eXéllIIination of int r acellu lar
fol d.t e.
The purpo5~ of thi s st u dy vas to da., elop a
chroaatography tecbnique and an extraction procedure for
Eolate vithin livin~ calis which could overco.e the probleas
( in previous studies. IJt:ll i z ing li PLC and the observatioD
19
1
(
(
t n.1 t the COn;UydSe dCtlYlty 1 n ra t VdS dctive
n dUt r ~ l pH (J 1), l baYe de'leL:>ped a technique "hlcn clppe~rs
t l) IJ ces er v e 1dOl1e Intrclcellular folates and .lnl.lzes
lfltdrconverslon -Jf tnese actlYe co.pouods durlnq extrdct.l.OD.
The 5 teps ln pcocessinq intracelluldc foldte wh 1 co l
a cl v e e.l cl. in ed ln cl U de:
:::onverSlOfl Jt t.) l at e t.J 01 Y q lu ta. a t e to
Il:)0 ù~ lu t a.at e
• T study .JE stdblllty of 5 t an d d r d t 0 l dt es ,jurlD-J
CJLjUydSe treat.eot
.II-effect -Jf cell dlsruption on folate dlstribut1..JD
l v- dlstr1.bution of folate ln noraal flbrobldsts usl.o'l
tad b~st of these procedures
I. ":ODverS1.0U of tolate po1ygluta.ate to Jaonoyluta.ate:
..:oaparlsion:lf two different pr ocedur es for conjuyase
tre,üaent and pr:ac1.pitation of protein (BSCT at pH 6.5 30d
lIethenol vs. HSCT at pH 5 and heat) de.onstrated that uS1.ng
aethanoi for preclpitation of protein was l es s ct es tr u c t 1. V e
ta Idblie folates (such as H4PteGlu and 10-CHO H4PteGlu)
th:sn VdS boill.ng. ID addit1.on to batter preservation of
ldu1.1e folate, intercon'lersion of forJaylated folates was
:11so less during this procedure as coapared to using HS CT
::1 t pH 5.
p r:> du ct s
The procedure using RSCT generated two breakdown ~
If h1cb vere id entified as brea kd olln prad ucts of
80
-, ' , l
1 1 J -CHU H4Pt aGlu.
tl a th p r ace :iu r B S '-Jaoerated soas <J, lO-CH2-H"PteGlù üy
da uilllnown aechanlsa which aiqht be due ta the presence Jt
tr.ice of foraaldehyde ln ODe of th~ aaterials.
therefar~ se le ct t:! i as cr vith preClpltdtlon Jt
J[)tl-!lUS wltb aethdllol dS th!::! standard procedur~ ta CODvalt
tJIY(Jolyyllltallat..; ta lIonoyluta.ate because lt catlsed t.:!SS
yenerdt ion of ~-CHO H4PteGlu thao dld the p [ oc e dure II 5 l U '-l
tiser dnd heat.
II-St~dy of stiblllty ot staudar ct f:) lat eS dur lny con 1u':fise
tredtlleot:
The decon1uqase procedure uSlnq rdt ssrua and aetn8u::>1
(Jerrllttad aore titan gOI racovery o t all folates tested
ex...:ept for lü-CHO A4PteGlu. All of the breakdown products of
10--':HO H4PteGlu dX::dpt PABGlu vere unlque ta thl.s fol.te 50
thdt the quant1ty af this foLite lD the orlg1ndl extrict
~ollld be estiœatej. Recoyer y 0 f t hese fol at es vas l.nt:erl.or
ta th1.3 vheo they vere eXiJosed ta huaao serua at pH 5 iUd
h~a t.
II 1- Eff ect of call disruptl.on aethads on folate
J .l::itr ibu tian:
( Tbree procedures for cell disruption vere investigat,d:
81
1
(
(
2) son1.cat1.on
.l ) f r e ez 1. n q d n d t h d Il 1.D q
r h ~ r e c 0 ver y J t l n t r ace l l Q l a l rad 1. 0 d c.""t l v l t Y Il d S li l /1 ù st
lldutL::al US1.ny the dlfferelit proc~ du res but t .. H:~S""
[J[,lceJures dff~cted the endoqeuous fol<lte d1.str1.butlon.
Jlrr~renra5 ln I,llates recovered t hese cell
11.j[u~t 1.on lIethods loci ud ed '1 rea te r l eco ver y
'10-..:tiU-fo1ate' jurlny extract1.oll v!.th heat ~han Il 1. th t be
Jtnsr procedures, yredter ':l8nerat1.ün of PlBGiu by SOn.l.Cdtl.JU
tèdÙ by the ùthdr procedures, an j ':1reater appearance of
'),10-Crl2-H4Pterau
T he sou rce
url!l 00110. l t l S
1 n ex t r a c~ \0 tee 11 s dis r-u pte d b Y f r e e Z 1 (} Y
of thisl add\ional 5,ll>-CH2-HoPteGlu ,.
upcob ab~_r.t it • d S pceseo t , n lbe
or111.0a1 ce11 dxtract and br::lKe jovo dur-ing extr-act1.on by
ù 0 11 i n '1, b ec au set heP A B G 1 u con t en t of extracts prepared by
b 0111. n-..} and freez1. n ':l-thall1.nq vere El[cess
'l,lù-CH2-H4PteGlu could have arisen b Y il n unknown /1 echan 1.5.
f rOll • 10-CHO f::llates' wh1.ch lI/ere present in 8xcess 111 the
bOlled e~tract, or- 5, 1 0 -C H 2 - H 4 Pte Glu present in the cells
d (} J p r- e S 8 r v ed in the frozen-thaved extr-act could have Daen
converted to ex:::ess IlO-CRO-folates' during extraction W.l.th
h eat. It is probable that the' 10-CHO-folates' or the
5,10-:d2-HqPteGlu, absent froa the cell extract prepared by
son1.c:ltion vere hydrolysed and recovered in the chroaatoyra.
82
-
1
J
, '
1 :i S t' A ~c.; lu.
,f NADP dni HAQPH t :> t tJ t! ce Il
,J[.)duced by fLeezlDq-thdvlD~ cdused aD UDPxpldlDf'd ::han.J~ lU
t n di!. S t [ lb ut l on ') t fol a tas. The r t! f or t:l
w hl l t:! t h~s t:! >:'DZ?, Iles
l D d ,,-t l '1 d t e ri l D the b 0 i l e deI t r ::i ct.
f.l[.)cedLlre fOl releaslny lDtract:dlular folate.
li haD ct:!lls 'ole re il srupted by son lcat l.on t IH:' prùport~->n
J f PA!j'; lu Il \ ~ Il l .J Il e r th aD Il l t h the o t h t! r pro c e dur es , Il !lI. C il
lodlcdtes .ore ieco.pos1t1on of foldte (hydrolysls of tlle
~-10 bond) dUrlD-l 50nlcat10n than "'hen ultracellula[ foldte.s
IIere reledsed "'l/tb nedt or freezl.ng-tbdw1ù-l.
IV- Dlstribution of folate 1n DOrllal fl.brobldst.s:
.ith the aboye obseryatl.OO fl.oal selection for ::a11
dItrlct10n and chrJ.atoyraphy procedure II~S :
1) boiling of cell suspension 1n phosphate butter pit
b.~ wlth O.2~ bata .arcaptoatbanol in a water bath for ~
Ill. n lJ tes tore le a se ln t r a ce 11 u l il r fol a t e
2) using RSCT pH 6.5 for conjugase treataent lod
precipitation of protein vlth lIethanol
3) HPLC for separation of folab(s
When this procedure vas applied ta study the
83
1 ~O.l..Jgenous foldte flbroblast -::onflueot
l 0:: ub i t ~ d Il l t h r 1 H ] Pt e r. lu, ha l f :J f the 1 il t r do ce li u l li r fol cl te
ln these cells IIde; in the fora of ')-CH H L& Pt.eGl u •
Tbe nell pr~::edure deaoostrdted less ')-CH3 H4 Ptd .. lu
tU:ln re ported fJrevlously (117), per h ap~:; beCduse
,)reservatlon of lab1le folate.
Toe observ.d [l1gh proportLon of ') - L HO H4 Pte Gl u l il t oe
r e p ~r t uSlnq lncubation Il lth hlladn sel ua at t:»r
le-'::ùn jugat Lon poly') IlItallld te fol d te (11 7) prùbably
d X IJ J.. ci 1 n e d b Y the .; b. e Il 1. C al c on ver s l. 0 n a t 1 l}- L tlU HI.lPteG lu to
)- ___ dO dl.lPteGlu under these conditions.
rhe dlstrlbut 10n of end ogenoas f 0101 te deSCrll.Jed h~re
1') tOi oorlldl hlIlidn tlDroblasts ln confluent yrovth II1thout
~LeV1JLlsly depleting the endoqenous folate of tbe cell.s.
rurth~r studles are requLrsd to desc[lb~ the d1.str1but1.on 1.0
::el15 ln loqarithllic grollth and in folate depleted cells.
The folate Il:Jnoqlutaaates cnnta.lned ln the eItract of
"'d.:iot:!d cells ara lSSQlllo:!d ta be iotracellular and nùt surf:lcl:!
b:Jund beCduse these cell5 lIere grovn l.n [3H] PteGlu 1I1l1..::h
r e ~ U1. ra deI po su r e t 0 ln t r a ca 11 u la ranz y 11 es to Eora these
falc1tes.
The capa=l.ty of h II Il an f 1. br 0 bl as t s tor grovt h 1.5
1111.ted. Therefore cells in this study vere liaited ta
those with fevar than 30 generations; .ainly bet veen 2)- 3D
generat ions.
,
J
BIBLIOGRAPH y
(l'12S)
. 2. w 1 l l s, L., B r l t. ~~ i. J. l, 1 0') y (1 'J 3 1 )
L II/ 1 l l s, L. il n j '; t I:i va [t, A., dr l t. J. E!x pt 1. ? a t tl 0 1. 1 tI, ... 4
.. • 0 d Y , P • L ., Lan ~ s ton, iii • C ., il Il d D d r b y , Il • J ., Prat' • S oc .
EXtitl. BiOl. "ed. l8,660(1938)
). Snt!ll,E.E., ilnj ~t~rson,II.H.,J. ddct[L~l.
3'1,2119 (1 (40)
o. l'tltchell,H. K., 5ne1l,E.E., anJ lil11~dlaS,R.J., J. AI.
Chet. Soc. 66,267(1944)
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