ADENYLATE CYCLASE I N
LM CELLS
Goltzman, D., Peytremann, A., Callahan, E., Tregear, G. W., & Potts, J. T., Jr. (1975), J . B i d . Chem. 250, 31993203. Hugli, T. E., & Erickson, B. W . (1977), Proc. Natl. Acad. Sci. U.S.A. 74, 1826-1830. Krishna, G., Weiss, B., & Brodie, B. B. (1968), J . Pharmacol. E x p . Ther. 163, 379-385. Marcus, R., & Aurbach, G. D. (1969), Endocrinology 85, 801 -8 I O . Marcus, R., & Aurbach, G. D. (1971), Biochim. Biophys. Acta 242, 41 0-42 1. Menegatti, E., Ferroni, R., Benassi, C. A., & Rocchi, R. (1977), Int. J . Pept. Protein Res. 10, 146-152. Merrifield, R. B. ( 1 969), Ad?).Enzymol. 32, 221 -296. Nicolaides, E. D., DeWald, H . A,, & Craft, M. V. (1963), J . Med. Chem. 6, 739 -741. Parsons, J. A,, Rafferty, B., Gray, D., Reit, B., Keutmann, H. T., Tregear, G. W., & Potts, J . T., Jr. (1975), in CalciumRegulating Hormones, Talmage, R. V., Owen, M., and Parsons, J . A,, Ed., Amsterdam, Excerpta Medica, p 33. Pdtthy, L., & Smith, E. L. (1975a), J . B i d . Chem. 250, 557- 564. Patthy, t.., & Smith, E. L. (1975b), J. Biol. Chem. 250, 565-569. Robinson, C. J . , Berryman, I., & Parsons, J. A. (1972), in Calcium, Parathyroid Hormone and the Calcitonins: Proceedings of the Fourth Parathyroid Conference, Talmage, R . V., and Munson, P. L., Ed., Amsterdam, Excerpta
VOL.
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Medica, p 5 15. Rogers, K., & Weber, B. H. (1977), Arch. Biochem. Biophys. 180, 19-25. Roosdorp, N., Wann, B., & Sjoholm, 1. (1977), J . Biol. Chem. 252, 3876-3880. Rosenblatt, M., Callahan, E. N., Mahaffey, J . E., Pont, A., & Potts, J . T., Jr. (1977), J . Biol. Chem. 252, 5847585 1. Rosenblatt, M., & Potts, J . T., Jr. (1977), Endocrine Res. Commun. 4, 115-133. Rosenblatt, M., Goltzman, D., Keutmann, H. T., Tregear, G. W., & Potts, J . T. Jr. (1976), J . Biol. Chem. 251, 159164. Rudinger, J. (1 97 I ) , in Drug Design, Vol. 11, Ariens, E. J., Ed., New York, N.Y., Academic Press, p 319. Sairam, M . R. (1976), Arch. Biochem. Biophys. 176, 197205. Tregear, G. W. ( 1 975), Pept., Proc. Eur. Pept. Symp., 13th, 1974, 177. Tregear, G . W., & Potts, J. T., Jr. (1975), Endocrine Res. Commun. 2, 561-570. Tregear, G. W., van Rietschoten, J., Greene, E., Niall, H. D., Keutmann, H. T., Parsons, J. A,, O’Riordan, J. L. H., & Potts, J. T., Jr. (l974), Hoppe-Seyler’s Z . Physiol. Chem. 355, 415-421. Tregear, G . W., van Rietschoten, J., Sauer, R. [T.], Niall, H. D., Keutmann, H. T., & Potts, J. T., Jr. (1977), Biochemistry 16, 2817-2823.
Effect of Membrane Phospholipid Compositional Changes on Adenylate Cyclase in LM Cells? Victor H. Engelhard,$ Michael Glaser, and Dan R. Storm*,§
ABSTRACT: Adenylate cyclase activities were examined in
mouse L M cell membranes which had been supplemented with polar head groups and/or fatty acids. Basal, fluoride-, and PGEl-stimulated activities varied systematically with changes in phospholipid composition, and PGEl-stimulated activities correlated with the average degree of unsaturation of the phospholipid fatty acids or with the primary amino group character of the phospolipid polar head groups. In addition, the K , for A T P of basal adenylate cyclase was systematically changed by both polar head group and fatty acid supplementation. Alteration of the membrane lipid composition also changed the temperature dependence of the enzyme and the
A d e n y l a t e cyclase is associated with the plasma membranes of a number of animal cells (Butcher et ai., 1965; Pohl et al., 1971a; Bilezikian & Aurbach, 1974; Engelhard et al., 1976a)
+ From the Department of Biocheniistr), University of Illinois, Urbana,
Illinois 61801. Rereiced February 22, 1978. This work was supported by National Science Foundation Grant PCM 73-001 245, Career Research Development Award AI00120-01, and a Grant from the Illinois Heart Association to D.R.S. and by National Institutes of Health Grant G M 21953 and Career Research Development Award G M 00193 to M.G. V.H.E. is a National Institutes of Health Predoctoral Trainee.
0006-2960/78/0417-3191$01.00/0
lag time between addition of PGEl and the onset of a change in catalytic rate. However, none of the alterations in the enzyme activity could be correlated with the viscosities of supplemented membranes and, instead, seemed to be characteristic for a specific polar head group or fatty acid composition. The data suggest a specific interaction of the enzyme with phospholipids and indicate that structural features of phospholipids may play a role in regulating adenylate cyclase activity. It is proposed that adenylate cyclase can exist in several different conformations in the membrane depending upon the phospholipid composition.
and has also been reported to be associated with particulate fractions of many other cell types. Several studies have implicated the importance of membrane lipids for adenylate cyclase activity and hormone stimulation. For example, membranes have been treated with nonionic detergents (Sutherland et al., 1962), digitonin (Pohl et al., 1971b), phospholipases Present address: Department of Biochemistry, Harvard University, Cambridge, Massachusetts 02 146. 8 Present address: Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98105.
0 1978 American Chemical Society
( P c l i l et al., 197 1b; Rubalcava & Rodbell, I973), and organic stilients (Rethy et a]., 1971). These treatments all resulted in z?:'Iiiges i n basal activity, hormone stimulation, or both. I n v:c:%ral studies, readdition of phospholipids to treated mcnilies has resulted in restoration of basal activity or hormone .-Il,iiulation to an extent that was dependent upon phospholipid ~ ~ ~ Jhead l d r group compositions (Pohl et al., 1971b; Levey. 197 I : ttctlij et al., 1972). However, the enzyme from different tissucs ii!treated wiith different agents exhibited varying requirements fui. phospholipids. This suggested that lo ,::-tivity might result from actions of these phospholipids other c!t:jii specific interactions with adenylate cyclase. There are t i b o uncertaintiea regarding the physical state of the added ;.iiospholipids and of the enzyme both before and after read!iiiioti of phospolipids. Several other experimental approaches have also been em;>!(ped to evaluate the importance of membrane lipids for srwt)'late cyclase activities. For example, Puchwein et al. re31 i t ed t he reversible in hi bi t i on of cat ec hola m i ne s t i m ul a t i on iden) late cyclase using filipin. which complexes cholesterol iclruein et al., 1974). Orly & Schramm have demonstrated i h t t the addition of free unsaturated fatty acids to erythrocyte liiL.nibranesenhanced isoproterenol stimulation of adenylate lase up to 25-fold (Orly & Schramni, 1975). In addition, mday et al. fused phospholipid vesicles with membranes and erved changes in both the temperature dependence and : ~ ~ i i v i of t y adenylate cyclase (Houslay et al., 1976). Z niethod which avoids some of the difficulties associated + ' i h reconstitution studies is the in vivo modification of the rwinbraiie lipid composition. Brivio-Haugland et al. observed ,i!:nificant changes in basal and hormone-stimulated aden) late se when rats were fed an essential fatty acid deficient diet ,io-Haugland et al., 1976). Techniques have now been s!i*wloped for the in vivo modification ofthe fatty acid or polar i!! nd group composition of mouse 1-M cells in tissue culture s .laser i et al., 1974; Williams et al.. 1974; Blank et al.. 1975; 1 .:iguson et al.. 1975; Schroeder et al., 1976). We recently I i ported the modification of adenylate cyclase activity in 1.M L', [Is as a result of the manipulation of the polar head group and f:i t) acid cornpositions (Engelhard et al., I976b). In this paper. ' . influence of membrane lipid compositional changes on nylatc cyclase and its responae to PGEI a r e extensively p.
t1,tterials and Methods .Marrrials. All chemicals were reagent grade. Organic solits were redistilled. Prostaglandin El (PCJEI)] was kind]), silpplied by Dr. John Pike, IJpjohn Co. ;rowth and Supplementation of Cells. Mouse LM cells e grown in suspension culture in Higuchi's medium (Hihi. 1970j containing 20 niM N-2-hydroxyethylpiperaz.2"-2-ethanesulfonic acid, pH 7.4, I g / l methylcellulose, 0 . 0 2 g / L sodium dextran sulfate. Growth and suppleIt'itioii \+ere carried out as previously described (Glaser et :, . 1973: Engelhard et al., 1976b). For polar head group pplementation. cells were centrifuged and resuspended in ;.:~.diiirncontaining 40 pg/mL. of the appropriate polar head !; uup in place of choline and were grown for 3 days ( 2 days for ' amino- I-butanol) prior to harvest. Fatty acid supplentation \vas carried out by addition of the fatty acid corned to bovine serum albumin to the growth medium. The t. 't! acid supplement was added at the indicated concentration i ?< ti before harvest. '.sij
Lipid Determinations. Lipids were extracted by the method of Bligh and Dyer as described by Ames (Arms. 1968). P h os p h o I i pid s were sepa r a t ed by t w0 -dimension 3 I t hi n- Ia 5 e r chromatography a5 previously described (Glaser et al., 197-2). Spots were visualized with 1 2 vapor, scraped, and eluted with 5 niL of CHC13:CH3OH:acetic acid:H20 (5:5:1 : I ) followed by 2 nil of C H 3 0 H . The extracts were combined, evaporated to dryness, and redissolved in CI-1C13:CH~01-I:1120 ( 2 2 :1 , 8 ) , The CHCI3 phase was used for total phosphate analysis according to the method of Ames (Ames, 1966). Fatty acid compositions were determined after extraction of the p h m pholipids by the method of Bligh and Dyer and separation of phospholipids from neutral lipids on a short unisil colurnii. Methyl esters were prepared in sodium inethoxidz iiiethanol (Applied Science) or concentrated HCI-methanol ( 1 :20)and chromatographed on 3 15% SP-2330. ('hromosorb PAWDMCS, 100-200 mesh column at 200 'C (Supelco). This colunin resolved cis and trans fatty acid isomers. Prcparution of Plostva Memhrrines. Plasma nieinbranes were prepared by a method previously described (Esko et al.. 1977). Exaniiriation of the adenylate cyclase activities i n the different fractions bhowed a similar purification to the N a + , Illine, 1 . l O , i bovine serum albumin, and 20 ink1 KzHP04, pH 7.5 (Salonion et a l . . 1973). I nig/nil. creatine kinase and 2 0 rnM phosphocreatine \+we rcgenerating aysteni. ATP iiseti in assii DE,AE-Sephadex A-2.5 chromatograph). AG-50 chromatography. Each sample contained 90 - 1 10 pg of membrane protein. Samples w r e incubated a t 30I. 80-I>-
-
5
60-
n w
2 3
40-
3
E $
1
20-
4O
1
do
$0
o:
BIO
io
IAO
;Ill
,Lo
NUMBER OF DOUBLE BONDS PER 100 FATTY A C I D S
FIGURE 2: Correlation between PGE1-stimulated activity and
number
of double bonds/100 fatty acids. Data were taken from Tables I and 111. The line was obtained by a linear least-squares fit.
anolamine shows the effect of successive removal of methyl groups from the choline nitrogen atom on adenylate cyclase activity. Removal of the first methyl group led to a 36% increase in basal and a 35% increase in PGEI-stimulated activities. Thus, no change in the degree of hormone stimulation was observed. Removal of the second methyl group resulted in no change in basal activity, but did cause an increase in PGEl-stimulated activity, such that an increase in hormone stimulation from 2.3- to 3.3-fold was observed. Removal of the last methyl group resulted in a doubling of basal activity with only a small increase in PGEl-stimulated activity. Consequently, the degree of hormone stimulation dropped back to the control level. Stimulation by PGEl G T P systematically decreased from 27.5- to 16.5-fold with successive removal of methyl groups from choline. Fluoride-stimulated activity was relatively unaffected by changes in polar head group composition, and, as a result, the degree of fluoride stimulation dropped from 12.4-fold in choline cells to 5.3-fold in ethanolamine-supplemented cells. The second series of polar head group analogues included ethanolamine, 3-aminopropanol, and 1-2-amino- 1-butanol, which all contain primary amino groups but modified alkyl chains. 3-Aminopropanol-supplemented cells were very similar
+
3 196
BI OCH EM I STRY
ENGELHARD. GLASER. AND STORM
80
‘zoor
/ I
YE
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a a
\ J S . ternatically with membrane microviscosity measured by fluorcsence polarization. Nor was there any apparent correlation between xtivation energies for enzyme activit!, hornione lap times and the activation energies for niernbrane micro or the, membrane viscosity that mily be predicted f r o m the phospholipid composition. The absence of a i ) ) correlation between enzyme activity and membrane fluidity measured by I ,6-diphenyl-l.3,5-hexatrienefluorescence polari7atiun inust be interpreted with some caution since the use of this d!c IC) deterniine the absolute value of the iiiembranc viscosity is subject to a number of uncertainties (bale et al.. 19'77: Kanato et al., 1977; Pessin et al., 1978). However, i t seems reasonable to conipare the relative changes i n thc rotational relawtion times of 1,6-diphenyl- 1 ,?J-hexatriene. or- the rnic.ro\i\co.litif, for similar membrane preparations. Thus i n cuiiclusion. i t appears that the variation of adenyhte cyclase aciii,itc arid hormone stimulation with the nieinbraric phosphr!lipid coniposition is due to direct o r indirect interactions w i t h p h o k pholipids rather than a general sensitivity tO bulk riieinbrar,e viscosity. Presumably, adenylate cyclase i n native rncnibranes can exist in several distinct conformations differing i n c;i tiil>tic ;tctivity which are dependent u p o n the lipid ciiviroiinicni Acknowledgiiient We arc grateful for the technical assistancc of t!iane T o s can0 and Nina Cohn. References Ames, B. N. (1966) M e t h o d s E n ~ ! ~ r mCy.l . 1 15. .4rnes, (3. G . (1968) ,J. B a ~ ~ t e r i o95. l . 833. .27har, S., Hajra, A . K,, & Menon. K.M .I. ( 1976) J . / j i o / . Chetii. 251, 7405. Rilezikian, J. P. R.. Aurhach. G . D. ( 1974) J . 13inl. I 'hctii 244, 157.
Rlarik, M. I
. f'iantadosi.
C ' . , fshaq. K . S.. Pr Sqrider. b'. ( I975)
Hiochetn. B i u p h y s , Res. C'or?irnuti. 6 2 , 983. Hocknert, J.. Roy, C., Rajerison. R.. Pr Jard. S . ( 1973) .I. Miol. C'hetki. 248, 5922. Brivio-Haugland. R P.. Lmis. S.L., Musch, k., IViildeck, 5 , . & Williams, 41. A . ( 1976) Biochiw. Bioph 150. Butcher, R. W.. Ho, R. S., Meng, El. C . & Sutherland, E. W . ( 1965) J . Bio/. Chem. 240, 45 15. Cuatrecasas, P. (1974) .Intiu. Ret. B i o r h e t ~ 43. . Ib9, Dale, E. D.. Chen. I.. A., &. Brand. I-. ( 1977) ./. Bioi. C'hrn? 252, 7500. Engelhard, V . H., Plut, I>. A,, & Storm, D. K ,( 1 976a) Biochinz. Bioplzys. .4cta 451* 48. Engelhard. V . Id., Esko, 1. I).. Storm. 1). R . , & (.ilaser, M . ( I 9 7 h b ) Pro(. :Vari. . k n d ,S'ri. (.'.S.:I. 73, 4482. Esko, J . D., Gilniore. R.. & Cilaser. M . ( 1977) Bioc,/zc/Tii,\irJ,
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