ANALYTIC4L BIOCHEMISTRY 55, 154-159 (1973)

Isolation of Rat Liver Plasma Membranes by Zonal

Centrifugation: Increased Yield with Ca”+

R. B. JONES, A. J. STATON, AND L. A. BIESOW

Experimental Medicine Division, Nnvnl Medico1 Research Imtitule, Bethesdn, Maryland 20014

Kewivrd February 14, 1973; accepted April 3, 1973

The application of zonal cenkifugation to t,he isolation of rat liver plasma membranes (1-4’1 has permit,ted some increase in yields over those obtained using more conventional techniques (5-8). In addition, mem- branes so isolated have been shown to be of at least’ comparable purity when judged by ultrastructural (11, or by enzymatic (2-4) criteria. Nevertheless, the yields by these techniques have remained on the orclcr of 5-1OyG,, much of the membrane fraction being lost in the early steps of differential centxifugation (3).

Recently, Ray (9) clemonstrat~ed that the recovery of membranes could be substantially improved by including Ca”+ in the homogeniza- tion medium, and by employing greater dilutions. However, the pro- cedure he used VW a modification of that of n’eville (5) which employs fixed-angle and swinging huckct, rotors. Under these conditions, it has been showy that CR?+ also increases the degree of contamination of iso- lated membranw by other cell organelles (8,101, and therefore additional purification is often necessary.

Because of the large volumes which it can accommodate. its long path length, and its capacity for dynamic unloading, the zonal rotor reduces this kind of contamination, and, as described here, permits the isolation of relatively pm-c membranes in good yields. Ftu%hermore, the pro- cedure is reasonably rapid and simple, thereby minimizing the time re- quired for the handling of membranw.

METHODS

Plasma membranes were isolatccl from the livers of male Spraguc- Dawlcy rats weighing 180-220 g by a procedure based on that of Ander- son et aE. (2). The animals mere maintained on water and Purina chow nd Zibitirm. Sacrifice was by clwapitation followed by in- sitar perfurion of the liver with ire cold 0.15 M saline. All subsequent procedures were carried out at 0-4°C. The livers were passed through a tissue press

154 Copyright @ 1973 by Arademic Press, Inc. ,411 rights of reproduction in any form rewwrci.

(Wnrvnrcl Apparatus Co., Nillis, Mass.)) and 6-8 g homogenized in 40 ml of 0.5 mix &Cl, and 1 m&I NaHCC),,, pH 7.6 (Cay- medium) with 15 strokes of a Dounce homogenizer, loose pestle. The homogenate was p:lssecl through four laycre of cheesecloth, and diluted with the C’a’:+ medium to a final volume of 30 ml/g of liver. This suhpenaiott was thn centrifuged at, 3400 rpm for 15 mitt in a HB-4 swinging bucket rotor of a RC-2 refrigerated centrifuge (Ivan Sorvall, Inc.. Norwalk, Corm) and the sulwrnatnnt fluid discarde(1. The pcllcts were resuspended in 40 ml of tlt(l C:L” meclium (Douttw homogcttizw, loose lwstle. IO strokea), diluted to 160 ml with the same solution, and reccntrifuged. The super- nntnnt fluid was again discarded, and t,ltc pelletJi; re~uspcnded in 40 ml of the Ca’+ medium by gentle homogenization. This suspension was then diluted to 160 ml with 1 tnx NaHCO,, pH 7.6, ant1 uwl as the sample for zonnl centrifugation. The zon:tl wntrifugation, am1 recovery of the membranes was carried out as dcscril~ed 118 Pflcgtfr et 01. (1).

The membrane pellets thus obtained were resuspentlcrl in :i wall rolumc of 1 111x1 NaHCO,, pH 7.6, rapidly frozen, and stored at -20°C.

Enzyme assays were carried out in a final volrunc~ of 2.0 ml under the c~ontlitions given in the following references: glucose-6-phosphatase (11)) acid pltospltat:tse (11 i , ant1 5’-ttucleotidaec (12) These canzyme activities were dctermincd based on phosphate rcleaee. The reactions were stolywd hy the nclclition of 2 ml of 1O~~C~ tricltloro:tcctic acid, and inorganic pltoe- pllnt~e was deterntin~tl (13) in :I O.&ml aliquot. of the supernalant. h control for nonspecific phospltataw act.ivity 114) with 1~hcnyI l)hoaphate as auhbtrate was included in the 5’-nncleotidasc assay.

Buccinate dehydrog~nnse :ictivit,y n-as determined (15 i based on a measured value of E = 7.40 cmy/~~ntolc for red1tqed 2- (p-nitropltcnyl) -.5- l~ltcn~ltet~rnzoliuni chloride at a wawlcngtlt of 490 nm.

Protein conccntrntions were determined by the method of Lowry et al. (161. ~Icnlhr:tne pellets xere fise(1 in 2% aqueous oso,, and stained with urnnyl acetate and Reynolds lead citrate for clcctron microscopy. S;cction\ of 600 A n-crc ~snniined.

So that, 5’-nucleotidasc activities could he used for calculating recover- ies, it was first demonstrated that observed incrrnses in epwific activity reflected increases in enzyme conccntrntions, rather than removal of in- hibitors. This was done hy diluting membrane preparations with either homogenate or the Ca” medium prior to the measurement of the enzyme nctirity. The presence of ltomogcnat’e was found to have no effect on the mcnsured activity.

TBBL

E 1

-* g 8p

ecifi

c Ac

t.ivit

,ien

of

Helen

ted

Enzy

mes

in

M

embr

sncs

an

d W

hole

Liv

er

Hom

ogen

at,e

sa

!z

l’rep

arat

icm

6’-N

ucle

otid

ase

Pucc

ilmte

de

hydr

ogen

ase

Acid

ph

osph

atas

e Ci

luco

se-6

-pho

spha

t,ase

8

-___

-_

_-

---

~__~

__

- __

- __

- __

_-

~Iem

hran

es

46.S

f

13.7

(S

) y”

0.05

7 *

0.02

3 (3

) 0.

043

* 0.

035

(3)

0.47

3 +

0.02

7 (3

) Ho

mog

enat

e 1.

32

* 0.

40

(S)

r” 3.

28

k 1.

69

13)

1.17

f

0.54

(3

) 2.

143

i 0.

064

(3)

_(

R at

,io

36.7

+

6.9

(S)

0.01

x *

0.00

3 (3

) 0.

043

* 0.

005

(2)

L-

0.22

0 f

0.00

s (3

) Ls

0

All

enzy

me

activ

ities

are

expr

esse

d in

M

mole

s of

pr

oduc

t pr

oduc

ed

/hr/m

g of

pr

otein

. Va

lues

ar

e giv

en

as

the

rne~

n +

the

stand

ard

w

devia

tion

with

th

e tllu

nber

of

de

term

inat

ions

iu

pa

rent

hese

s.

Each

tle

t~er

min

at,io

n re

pres

ents

a se

para

te

mem

brar

le pr

epar

ation

. Th

e 2

ratio

giv

e11

is t

hat

of

the

spec

ific

enzy

me

:wtiv

ity

in

each

m

eml)r

ane

prep

xmtio

u to

th

at

in

thp

hont

ogen

nte

from

wh

icah

it wa

s pr

epar

ed.

z

EOLATION OF RAT LIYER MERIBRANKS 157

The average yield of membranes isolated by the above procedure was equivalent to 1.31 -t- .25 mg of membrane protein/gm wet weight of liver,l representing 37.5 k 8.9% of the total 5’-nucleotidase activity of the initial hom0genate.l These values are similar to those reported by Ray (9).

The purity of the membrane preparations was evaluated by dctermin- ing the specific activities of several marker enzymes in t,he membranes relative to their activities in the initial homogenates (Table 1). The specific activity of 5’-nucleotidase is coml)arable to that reported for membranes isolated by ot.licr proccdurcs, while the nctivit.ies of suc- cinate debydrogcnnsc, acid phosphataee, and glucose-6-phosphata+c (rep- resenting potential contaminants) are substantially lower (2.3,6,8-101. That such differences are valid and not a result of discrepancies in methodology is indicated by the specific activity ratios (Table 1). In addition, any possible effects of freezing and storage at -20°C on the activities of these enzymes was evaluated, and found to be negligible under the conditions employed (storage from one to three days).

As a further check on the purity of the membrane preparations, they were evaluated by electron microscopy. This revealed characteristic membrane structures (Fig. I), but no recogniznblc nuclei, mitorhondria, or lysosomes.

While some acid phosphatase is present in the membrane preparations, it is doubtful that it represents contamination by intact lyeosomes. The homogenization medium would be espect,ecl to completely disrupt. lyso- somes by virtue of its hypotonicitp (17)) and in fact, repeated freezing and thawing of the homogenates (time, s 15) resulted in no further in- crease in t’heir acid phosphatase activity. This obrervntion confirms the nbsencc of intact lysoson~es in the initial homogenatce (17) and, there- fore, in the membranes, but leaves unnnswerctd nhcther the activity in the membrane preparation is intrinsic, or an ndsort)ctl contaminant.

These studies reemphasize the import#ance of Ca’+ in maintaining the structural integrity of plasma membranes. How-cvcr, it would seem that it is primarily during the mechanical stress of homogenizat,ion t’hnt Ca”+ exerts it.s beneficial effcrt.s. Based on the data not. shown hcrc. the omis- sion of Ca’+ from the homogenization medium resulted in low yiclcls. On the other hand, the addition of Ca”+ to the dcnsitp gradient did not affect the yield, but, did result in membranes that were hcavilp contaminated with other subcellular orgnnelles. Because of the large volume which the zonal rotor can accommodat,e, thcrc is conklerahle diMion of the Ca’+ during gradient centrifugation, and it, is probably thip feature

’ Mean zk stnndnrd error for 8 different membrane preparations

JONES, STATON, AND KTESOW

more than any other which permits the isolation of relatively clean membranes.

SUMMARY

Rat liver plasma mrmbranes ww isolated by zonal centrifugation after homogenization of the livers in a Ca”+ containing medium. The yield was 37.5cjc, based on the recovery of 5’-nucleotidase. The membrane purity as judged enzymatically and by electron microscopy was superior to that reported for other procedures.

~~~:IiNOWJ,EDC;h,lE?u’TS

Tlic autlior;; wish to thank Drs. I,. G. Dickson and A. E. McKee. Jr. of tbc

Extxrimcntnl Pathology Division. Naval Medical Rcsenrch Institute. Bethesda, Maryland for t,lrls preparation and interpretation of the clwtron mici~ogrnplrs.

This work was supported by the Bureau of hlcdicine and Surgrry, Sax‘>- Ikpart- ment Rcerarch Suhtask MR041.20.01.0399. The opinions and stntcmcnts contninrd herein arc tlrc> prixatc ones of the authors and arc not lo lie c~owlrucd a:: ofIici:rl or rctlecting tlrr, vitas of the Kax-y Depart mrnl or of thr Kaval Scr\ icr at I,argc.

l~EFI!!RENCES

1. PI~I,IZW:R. Ii. c'., ,1XDKRSON. s. (;.. :\ND SSYDER. li‘. (1968) .!%Oc'iiC'V&i~ 7, 2826. 2. AND~:RSO~, S. G.. LANBIKG, 8. I., LICHCRXW. I., I<.~YKKIN, c. T., ASD EI.ROD, H.

(1966) irk “Biological Properties of the hlammalian Surface Mcmbrnnc” CL. il. Mason. cd.). p. 23. Wistar Institute Prcas, Philndrlt~hia.

3. &bmx~, R. A.. ASD BOYLE, W. (1969) &ochinl. ,%o$q/s. Actn 17’3, 377. 4. EVANS, FY. II. (1970) &ochena. J. 166, S33. 5. NEWLE. D. hl., JR. (1960) J. Biophp. Lliochem. Q/lo/. 8, -113. 6. EMRZEU)T. I’.. Bus. C. J., BEX~ETTI, IX. I,.. AND Ki'xm. P. H. (1964) Kophgs.

Acfa 90, 126. i. LANsIsu. A. 1.. UELKHOD&. RI. L., I.TN(‘II. W:. E., AND I,IFXERM.\N, I. (1967)

J. Biol. Chem. 242, 1772. 8. HAWKINS, G. F.. AND JAW~Z, J. il. (1972) A/ur/. Biochcm. 49, 290.

9. RAY. ‘I.. I<. (1970) Riochirn. Bioph~s. Acfrr 196, 1. 10. EMMELOT, P., ASD Ros, C'. J. (1969) IIL~. J. Cn~er 4, 705. 11. HUIMXIER. G., AND m'wr, G. (1965) LYcrt~tre (Lorcdon) 205, 799. 12. MICHELL, R. H., AFD HAWTHORNE, J. N. (1965) Biochcnl. Biophys. I<~,s. CC,,~~~~~~,~.

21, 333. 13. GOLDENBERG, H., AND FERNANDEZ, 8. (1966) C’lin. Clwm. 12, 871. 14. I+JW.SRDS, M. J.. ASD MAGUIRE. M. H. (1970) l\Iol. P/~r/wwxl. (3, 641.

15. PENKINGTON. R. J. (1961) Biochem. J. 80, 649. 16. I~OWW. 0. H.. HXWEHROUGH, N. J.. FARK A. I,.. .\SD H.\NDAI,L. R. J. (1951) J.

Bio2. Chcm. 193, 265.

17. GIANETTO, R.. AXD DF: Duvs, G. (19.55) Biochrm. J. 59, 133.