Phospholipids, sterol carrier protein 2 and adrenal steroidogenesis

Biochimicu et Biophysrca Acta 834 (I 985) 324--330

324

Elsevier

BBA 51903

Phospholipids, sterol carrier protein 2 and adrenal steroidogenesis George V. Vahouny a**,Ronald Chanderbhan ‘, Phylene Stewart ‘, Robert Tombes a, Eru Keyeyune-Nyombi ‘I, Gary Fiskum a and Terence J. Scallen ’ a Department of Biochemistry, The George ~ushjngto~ lJniuersi(v, School of medicine und Health Scrence. 2300 @e Street. Northwest ~ushjngto~, D. C. 2037, and’ Department of Bi~hemist~, University ofNewMexico, Schoot o~~edi~ine, Aibu~er~ue~ NM (U.S.A.) (Received

Key words:

Steroidogenesis;

Phospholipid;

January

Sth, 1985)

Sterol carrier

protein;

Cholesterol;

(Rat adrenal

cell)

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein, (SCP,) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of p~sphatidylc~line, phosphatidylin~itol, or the 4’-phosphate aud the ~,5~~iphos~ate derivatives of ph~phatidylinositol were without effect. With the adrenal plasma membrane prepa~tion, none of the added ph~ph~ipi~ affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact rnit~hon~~ only cardiolipin, among the various ph~pholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 PM), the stimulatory effect was only half that observed with 0.7 PM SCP,, and the two effecters were not synergistic. SCP, caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-4!&,, had no effect on distribution of mitochondrial membrane cholesterol.

Introduction Studies by Farese and colleagues [l-4] have implicated polyphospho~lated phospholipids as mediators of cholesterol utilization in steroidogenie tissues. In addition to reporting rapid turnover of these lipids during hormonal stimulation of steroidogenesis [3], it has also been reported that addition of phosphatidylinositol, or the 4’-phosphate analogue, will stimulate corticosterone production by dispersed adrenal cells [3], and that cardiolipin, in particular, can influence ~t~hond~al pregnenolone production [l]. * To whom correspondence should be addressed. Abbreviation: ACTH, adrenocorticotropin. 0005-2760/85/$03.30

0 1985 Elsevier Science Publishers

The latter finding relates to reports [5-71 of the importance of phospholipids for activities of enzymes of the inner mit~hondrial membrane. and to the possible role of polar lipids in adrenal cytochrome

P-450,,,

activity

[8,9].

We have reported that preparations of homogeneous sterol carrier protein, (SCP,) can function to solubilize cholesterol from adrenal lipid inclusion droplets and deliver this substrate to mitochondria for pregnenolone synthesis [lo]. Furthermore, homogeneous SCP,, free of any lipids, will increase utilization of mitochondria cholesterol. This occurs even with mitochondria obtained from adrenals of rats treated with cyclohe~mide, during which cholesterol accumulates at the mitochondrial outer membranes [lo]. Since SCP,

B.V. (Biomedical

Division)

325

does not directly influence the interaction of cholesterol with ~t~hond~~ cytochrome P-450, [ll], the stimulation of cholesterol utilization by mitochondria appears to be a function of SCP,-dependent translocation of sterol to the inner membrane site where cytochrome P-450 is localized [11,12]. In the present study we have evaluated the relative effects of several polar phospholipids on specific parameters of adrenal steroidogenesis. These included effects on corticosterone output by dispersed adrenal cells, on plasma membrane CAMP production, and on ~tochond~al pregnenolone synthesis. In this latter model, comparative studies with cardiolipin and SCP, have also been conducted in an attempt to distinguish the site of action of these effecters. Experimental procedures

Adrenocortical cells were prepared from unstimulated rats (Charles Rivers, Wilmington, MA), and were characterized as described earlier [13]. These were dispersed in Krebs bicarbonate buffer (pH 7.4)/11 mM ~ucose/O.O8 mM bovine serum albumin/765 mM Ca”. Aliquots of the suspension (1.9 ml containing (2-5). 105 cells) were incubated at 37°C for 30-60 min following addition of 0.1 ml of ACTH,_3, (0.7 nM), or the appropriate phospholipids preparation (200 PM). The phospholipids (Sigma Chemical Co., St. Louis, MO) were dispersed as described by Farese and Sabir [l] and Tanaka and Strauss [14]. These were dissolved in propylene glycol and sonicated in the above Krebs buffer immediately before use. corticosterone was determined ~uoromet~cally 1151and by radioimmunoassay (Radioassay systems, Carson, CA). Rat adrenal membranes were prepared by a modification of the procedures of Londos and Rodbell [16] and Dazord et al. [17]. Incubations contained the following components in 450 ~1: 0.2 mM [n- 32PJATP (ICN Chemical and Radioisotope Div., Irvine, CA) (5 mM Mg*+)/l mM CAMP/IO FM GTP/l mM dithiothreitol/O.l% bovine serum albumin/2.5 units creatine phospho~nase/S mM phosphocreatine/ 1 mM 3-isobutyl-l-methylxant~ne/lO g membrane protein/l25 mM TrisHCl buffer (pH 7.6). Where indicated, additions

were made in 50 ,ul buffer and included 10 mM NaF, 2.5 - lo-’ M ACTH,_,, or the phospholipids (200 PM). For these studies, phospholipids were dissolved in propylene glycol and sonicated in 0.15 M K&Cl/50 mM Tris-HCl buffer (pH 7.6) [14]. The reaction was carried out for 15 min at 30°C and was concluded by addition of 100 ~1 of a solution containing 2% sodium dodecylsulfate, 45 mM ATP and 1.3 mM CAMP. After addition of [3HJcAMP as internal standard, samples were boiled for 3 min, cooled, and diluted with 1 ml distilled water. Labeled CAMP was separated from other nucleotides by the method of Salomon et al. 1181. This involved initial cation exchange chromatography on Dowex AG 5OW X 4 (Bio-Rad) and subsequent chromatography on Alumina WN-3 (Sigma). Fractions obtained by elution with 0.1 M imidazole-HCl buffer (pH 7.5) were collected directly into scintillation vials. These adrenal plasma membrane preparations were comparable to those reported by others 116,171, with respect to effects of EGTA and Ca2+, optimal concentrations of cofactors, and responsiveness to ACTH and NaF. It was determined that maximal cAMP production was obtained at an ACTH concentration of 2.5 PM, and half-maximal response was at 0.25 PM. These concentrations are also comparable with those reported by others [16,19,20]. Adrenal mitochondria were prepared as described earlier [lO,llJ. Pooled adrenals from up to 50 rats were homogenized in 4 ml cold 250 mM sucrose/l mM EDTA/20 mM nicotinamide/lfi mM Tris buffer (pH 7.4) [21]. The homogenate was centrifuged for 10 min at 800 x g to remove cell debris. The sediment was resuspended in 8.0 ml of 250 mM sucrose and centrifuged at 800 x g for 10 min. The pooled supernatants were centrifuged at 5000 X g for 10 min at 4°C. The resulting mitochondrial pellet was resuspended in pH 7.5 buffer [22] consisting of the following components: 36 mM Tris (pH 7.5)/10.4 mM sodium phosphate/39 mM succinate/5.2 mM magnesium chloride/O.25 M sucrose. Protein was determined on all preparations by the method of Lowry et al. [23]. Incubation flasks contained 1 ml mitochondrial suspension (l-2 mg protein) and 1 ml buffer containing either the phospholipid suspension or SCP, [lo,11 J, at concentrations indicated for indi-

326

vidual studies. Pregnenolone was determined by radioimmunoassay [ll]. For studies on membrane distribution of mitochondrial cholesterol, the mitochondria were prepared from adrenals of 45 rats given cycloheximide (10 mg) intraperitoneally, 60 min prior to killing. These were incubated for 30 min at 37°C in media containing 0.75 mM aminoglutethimide alone, or with added cardiolipin (200 PM) or SCP, (0.7 PM). Mitochondria were reisolated by centrifugation at 5000 x g for 10 min and were resuspended in hypoosmotic buffer consisting of 20 mM phosphate buffer/0.2% bovine serum albumin adjusted to pH 7.4 [24,25]. This was maintained on ice for 20 min, and centrifuged at 17000 X g for 30 min at 4O’C. The pelleted membrane fraction was resuspended in 0.25 M sucrose and layered over 3.75 vol. of 7.5% (w/v) Ficoll in 0.25 M sucrose [28]. This was centrifuged at 10 000 X g at 4’C, and separated into four fractions [25]. The upper turbid fraction (fraction 1) consists of outer membranes, and the pellet (fraction 4) represents mitoplasts and intact mitochondria. The composition of the intermediate fractions have also been described by Privalle et al. [25]. With the disruption and fractionation procedure employed in the present studies, the membrane pellet, following hypoosmotic disruption, containing 87% of the total mitochondrial protein. Of this, 10.1 rt 1.4% was associated with the outer membrane fraction, and 56.0 + 4.0% was in the mitoplast fraction. These distributions are comparable to those reported earlier [24,25]. Purity of the fractions was determined by assay of the distribution of rotenone-insensitive NADH-cytochrome reductase as an outer membrane marker [27], and succinate dehydrogenase as a marker for inner membranes [28]. Based on these criteria, the outer membrane fraction was 9% contaminated with inner membrane, and the mitoplast fraction was 12-13% contaminated by outer membranes. These results are also comparable to those reported earlie [24,25]. Cholesterol content of all Ficoll fractions was determined by gas-liquid chromatography as described earlier [lO,ll]. Results

Addition of liposomal dispersions phosphatidylcholine, phosphatidylinositol,

of

either or the

TABLE

I

EFFECT OF LIPOSOMAL PHOSPHOLIPIDS ON CORTICOSTERONE PRODUCTION BY RAT ADRENAL CELLS Rat adrenocortical cells, in a final volume of 2 ml (average cell count, (2-2.5).105 cells/ml; average viability 97%). were incubated for 60 min in the absence or presence of 0.71 nM ACTH or 200 pM phospholipids dispersed as described by Farese and Sabir [l]. Results represent means of four values for net corticosterone production f SE. Additions

Net corticosterone production (2.S.105/ng per 60 min)

ACTH Phosphatidylcholine Phosphatidylinositol Phosphatidylinositol 4’-phosphate Phosphatidylinositol 4’,5’-diphosphate Cardiolipin

213.0 f 27.0 3.8 * 15.2 0.0 i: 15.2 7.6 + 22.8 - 22.8 f 19.0 41.8+27.0

4’-phosphate or 4’,5’-diphosphate derivatives of phosphatidylinositol, had no significant effect on steroidogenesis by rat adrenal fasiculata cells (Table I). Addition of cardiolipin (200 PM) caused a variable but significant increase in corticosterone output, but this was not further enhanced by increasing the concentration of the phospholipid. With adrenal plasma membrane preparations, baseline levels of CAMP production were low (3.6

TABLE

II

EFFECTS OF ACTH AND PHOSPHOLIPIDS ON cAMP PRODUCTION BY RAT ADRENAL MEMBRANES A rat adrenal membrane preparation (10 ng protein) [16.17] was incubated with 200 nM dispersions of the indicated phospholipids in the absence or presence of 0.25 nM ACTH,_L, (total vol. 0.5 ml) for 15 min at 30°C. Production [32P]cAMP from [ a-“PJATP was assayed by the method of Salomon et al. [lS]. Results are means + S.E. for three determinations. Additions

None Phosphatidylcholine Phosphatidylinositol Phosphatidylinositol4’-phosphate Phosphatidylinositol4’.5’-diphosphate Cardiolipin

Net CAMP production (pmol/min per mg) - ACTH

+ ACTH

3.6rtO.2 4.9k1.6 3.7 * 0.1 4.3kO.l 4.2 + 0.7 4.2k1.7

11.9il.l 10.5 + 1.8 12.0i1.3 10.5 + 0.2 10.7 f 0.6 9.2k1.7

327 300

TABLE III

1

EFFECT OF PHOSPHOLIPIDS ON PREGNENOLONE PRODUCTION BY RAT ADRENAL MITOCHONDRIA Adrenal mitochondria (l-2 mg protein) from quiescent killed rats were incubated in a final vol. of 2 ml for 30 min at 37°C with phospholipid dispersions (200 PM) indicated below. Pregnenolone was determined by radioimmunoassay. Results are means f S.E. from three studies.

5004 400

300 200

Addition

Pregnenolone 100

None Phosphatidylcholine Phosphatidylinositol Phosphatidylinositol 4’-phosphate Phosphatidylinositol 4’,5’-phosphate Cardiolipin

ng/mg protein

% of control

26.6kl.6 24.7 f 0.3 26.1 It 0.8

100+6 93+1 98*3

25.8k1.6

97k6

26.6f0.3 48.4 f 1.6

100*1 182rt6

k 0.2 pmol/min per mg) and were not significantly altered by addition of any of the phospholipid dispersions (200 PM) (Table II). Addition of ACTH,_,,,at one-half maximal concentrations (0.25 PM), resulted in a 3.3-fold increase in CAMP production, and this was not altered significantly by further addition of any of the phospholipid preparations (Table II). The effects of individual phospholipid dispersions on baseline levels of mitochondrial pregnenolone production are summarized in Table III. Only cardiolipin (200 PM) produced a significant increase (182 & 6% for control) in pregnenolone output by adrenal mitochondria. This effect was concentration-dependent (Fig. 1) with half-maximal pregnenolone output occurring at about 120 PM cardiolipin. This increased utilization of endogenous mitochondrial cholesterol for pregnenolone synthesis was also enhanced by addition of homogeneous SCP,, but at much lower concentrations than was required with cardiolipin. In separate studies, mitochondrial pregnenolone production was assayed in the presence of each effector alone and in combination. Addition of 100 PM cardiolipin gave pregnenolone output of 140-3s of control, while 0.7 PM SCP, caused a 487 k 13% increase in pregnenolone production. When added together, the cardiolipin showed no stimulation over that observed with SCP, (453 f

i

CARDIOLIPIN.

,,M

STEROL

CARRIER

PROTEIN.

,,M

Fig. 1. Stimulation of mitochondrial pregnenolone synthesis by cardiolipin and sterol carrier protein,. Preparation of adrenal mitochondria and incubation conditions are described under Experimental procedures. Results represt means from three studies* S.E. and are expressed as percent of control (no additions). Baseline levels of pregnenolone determined by radioimmunoassay in individual studies varies from 26 to 50 ng/mg protein.

13%). However, at 200 FM cardiolipin (193 + 3%) the pregnenolone response was additive (567 f 16%) with that obtained with SCP, alone (418 + 4%). During studies on the distribution of cholesterol between mitochondrial outer and inner membrane preparations, there was a significant redistribution of membrane cholesterol during the 30 min incubation of mitochondria in vitro (Table IV). Thus,

TABLE IV DISTRIBUTION OF CHOLESTEROL BETWEEN OUTER AND INNER MEMBRANE FRACTIONS OF ADRENAL MITOCHONDRIA Adrenal mitochondria were prepared from 45 male rats given 10 mg cycloheximide 60 min prior to tissue acquisition. These were either disrupted immediately or were first incubated for 30 min at 37°C in the presence of 0.7 mM aminoglutethimide and the additions indicated. Methods for separation of membrane fractions and analyses of cholesterol and protein are described under Experimental procedures. Additions

None, preincubated None, incubated 30 min Cardiolipin (200 pM) SCP, (0.7 PM)

Cholesterol (pg/mg protein) Outer membrane

Mitoplast

14.6 8.8 10.1 6.5

5.2 9.7 9.2 13.1

328

in the present study, the cholesterol concentrations of the outer membrane and the mitoplast (inner membrane and matrix) fractions of mitochondria were similar, following a 30 min incubation of intact ~tochondria in the presence of aminoglutethimide which was added to prevent further utilization of the cholesterol. Incubation of mitochondria with 200 PM cardiolipin had no effect on the distribution of cholesterol between these membrane fractions (Table IV). while with SCP, (0.7 PM), there was a further significant shift in cholesterol to the rnitoplast fraction. Discussion Previous studies by Farese and colleagues [l-4] have provided strong evidence for the role of the phosphatidate-polyphosphatide pathway in the regulation of membrane-associated events in adrenal cortical tissue. Within minutes after acute ACTH treatment of rats [l] or of adrenal quarters in vitro [3f, there were significant increases in adrenal phosphatidic acid, phosphatidylinositol and polyphosphoinositides, but not in levels of phosphatidylcholine or cardiolipin. Incubations of dispersed adrenal cells with phosphatidylinositol [2] or its 4’-phosphate derivative [2,3] resulted in increased corticosterone output to levels about 25% of that observed with ACTH. Studies with adrenal mitochondria suggested that the 4’-phosphate and 4’,5’-diphosphate derivatives of phosphatidylinosito1 (11 as well as cardiolipin [1,3] enhanced pregnenolone production whe included at levels of 100-200 PM. These findings supported the suggestion [4] that specific glycerophospholipids might represent the effecters, found in cell cytosol, which by adrenal regulate cholesterol utilization mit~hond~a. Findings by others have, however, not been entirely consistent with this hypothesis. Tanaka and Strauss [14] were unable to duplicate the acute changes in the concentrations of phosphatidylinositol or its phosphate derivative during acute stimulation of ovarian tissue in vivo. Furthermore. using ~tochondria from rat corpora lutea, only cardiolipin among the various phospholipids tested, was able to stimulate pregnenolone production [14]. Igarashi and Kimura [29] found that ACTH treatment of dexamethasone-treated rats.

or incubation of cells with CAMP, resulted in increased levels in mitochondrial phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine. No changes were observed in other phospholipids~ including cardiolipin, or in the phospholipids of other membranes, including plasma membranes, endoplasmic reticulum and peroxisomes. The primary sites of phosphatidylinositide synthesis are the cellular membrane fractions [30,32], and a specific role of phosphatidylinositol in regulation of membrane adenylate cyclase has been suggested [33-351. Furthermore, both cardiolipin [8] and phosphatidylinositol [9] have been reported to be activators of cytochrome P450,,,, and to increase the affinity of the enzyme for cholesterol in isolated model systems. Whether this cholesterol is already associated with the mitochondrial inner membrane, or that the polar lipids can modulate membrane redistribution of cholesterol has not been previously tested. The present study was designed to test certain aspects of the effects of specific phosphoiipids on responses by intact adrenal cells, and adrenal plasma membrane preparation and adrenal mitochondria. The adrenocortical cell preparation contains concentrations of endogenous cholesteryl esters and phospholipids comparable to those found in the intact tissue [13]. Without unusual care in animal handling and cell preparation, there is extensive hydrolysis of the endogenous esterified cholesterol. and the subsequent acute corticosterone response to ACTH or CAMP is increased [19]. This preparation has also been characterized with respect to various aspects of cholesteryl ester metabolism and arachidonate metabolism [13,36,37]. Incubation of these cells with liposomal dispersions of phosphatidylchoiine, phosphatidylinositol or the phosphate derivatives of phosphatidylinosito1 did not result in increased corticosterone output. However, cardiolipin addition did cause a variable but significant increase in cellular corticosterone output to a level about 20% of that obtained with ACTH. Based on various pieces of evidence, the steroidogenic response of adrenocortical cells to ACTH appears to be mediated via the CAMP cascade. TO test this aspect of phospholipid responses, we have

329

employed an adrenal membrane preparation which has been characterized according to earlier reports [15,17,19,20]. The adenylate cyclase of these membranes is directly activated by NaF, and retains ACTH sensitivity at hormone levels previously reported to be maximally effective [l&19,20]. With these membranes, none of the phospholipid dispersions affected adenylate cyclase directly nor did they modify ACTH-dependent CAMP formation. it is possible, however, that unless catalyzed by non-specific lipid transfer proteins, the addition of these phospholipid dispersions alone might not directly alter the composition or fluidity of the membranes sufficiently to elicit either stimulatory or inhibitory responses of the adenylate cyclase 1351. Perhaps of the greatest significance is the suggestion that specific glycerophospholipids could represent important effecters of mitochondrial pregnenolone synthesis during stimulation of mitochondria by addition of cytosolic fractions [4]. Others have reported a stimulator effect of adrenal cytosol proteins [1,38-411, and this has been attributed to specific cytosolic proteins [1,38,39], to the cholesterol content of cytosol [41], and to specific polyphosphorylated phospholipids 1421. In this latter connection, Farese and colleagues [2,3] reported that additions of cardiolipin, or of the 4’-phosphate or 4’,5’-diphosphate derivatives of phosphatidylinositol at levels of lo-200 PM, induced remarkable increases in mitochondrial pregnenolone output, In the studies of Tanaka and Strauss (141 and in the present study, only the liposomal cardiolipin preparation was able to elicit an increased pregnenolone response by isolated mitochondria. Although this polar lipid has been reported to be an activator of mitochondrial cytochrome P-450,, , its cellular [43] or mitochondrial con~ntrations are not altered during ACTH stimulation in vivo or in vitro. Nevertheless, the possibility exists that membrane redistribution of this phospholipid might occur in response to ACTH, and that this could affect either the activity of the side-chain cleavage system, or the transfer of ~t~hondrial membrane cholesterol to the cytochrome P-450,,, , located on the inner face of the mitochondrial inner membrane [43]. At present, there are reports of two polypeptides, found in adrenal cytosolic preparations,

which can stimulate pregnenolone production by adrenal mitochondria. The smaller of these polypeptides (‘2.2 kDa peptide’) may represent the labile protein factor induced by ACTH [44]. This peptide has not been shown to transfer cholesterol to the mitochondria, but does appear to affect interaction of cholesterol with mitochondrial cytochrome P-450,,, [43] via an as yet undetermined mechanism. A second polypeptide sterol carrier protein, (SCP,) has also been reported to transfer cholesterol from adrenal lipid droplets to mitochondria [lo] and to stimulate mitochondrial pregnenolone production [ll] by a mechanism involving redistribution of cholesterol from outer to inner mitochondrial membranes [12]. This protein, however, appears to have no direct effect on the cytochrome P-450,,-cholesterol interaction [ 111. Treatment of adrenal cytosolic preparations with a specific anti-SCP, IgG results in complete loss of the stimulatory effect of cytosol on mitochondrial pregnenolone synthesis [44]. However, it is not yet known whether SCP, levels, or turnover, are affected by ACTH, nor have studies yet been conducted to assess the relationship between SCP, and the 2.2 kDA protein. A comparison of the effects of cardiolipin and SCP, on pregnenolone production by adrenal mitochondria, and on distribution of ~t~hon~al membrane cholesterol, clearly demonstrated that SCP,, but not cardiolipin, can affect transfer of cholesterol from outer to inner mitochondrial membranes in mitochondria, which are unable to further utilize the chol~terol for pre~enolone synthesis. The stimulatory effect of cardiolipin on mitochondrial pregnenolone synthesis, in contrast, appears to be directed at a different site, which is most likely the interaction of cytochrome P-450s~~ and cholesterol [8]. Since phosphatidy~nositol also appears to affect this latter reaction, it seems likely that the transfer of cholesterol to mitochondria, and from the mitochondrial outer membrane to the inner membrane site of cytochrome P-450s~~ is a function of one or more polypeptides. The activity of the cholesterol side-chain cleavage enzyme to further utilize this cholesterol appears to be, at least in part, regulated by the polar phosphohpids. Whether either or both of the polypeptides also affects membrane distribution of the polar lipids remains to be determined.

330

Acknowledgements This work was supported by United States Public Health Service Grants AM-32309 (G.V.V.), CA-32964 (G.F.), HL-16796 (T.J.S.) and AM10628 (T.J.S.). References 1 Farese, R.V. and Sabir. A.M. (1979) B&him. Biophys. Acta 575. 299-304 2 Farese, R.V., Sabir, M.A., Vandor, S.L. and Larson, R.E. (1980) J. Biol. Chem. 255, 5728-5734 3 Farese, R.V., Sabir, M.A. and Larson, R.E. (1980) J. Biol. Chem. 255, 7232-7237 4 Farese, R.V. (1983) Metabolism 32, 628-641 Biophys. Res. 5 Fry, M. and Green, D.E. (1980) B&hem. Commun. 93, 1238-1236 6 Fry, M. and Green, D.E. (1981) J. Biol. Chem. 256, 1874-1880 7 Vik, S.B.. Georgevich, G. and Capaldi. R.A. (1981) Proc. Natl. Acad. Sci. USA 78, 1456-1460 8 Lambeth, J.D. (1981) J. Biol. Chem. 256. 4756-4762 9 Kowluru, R.A., George, R. and Jefcoate, C.R. (1983) J. Biol. Chem. 258, 8053-8059 R., Noland, B.J., Scallen, T.J. and Vahouny. 10 Chanderbhan, G.V. (1982) J. Biol. Chem. 257, 8928-8934 R., Noland, B.J., Irwin, D.. 11 Vahouny, G.V., Chanderbhan, Dennis, P., Lambeth. J.D. and Scallen, T.J. (1983) J. Biol. Chem. 258, 11731-11737 R., Fiscum, G.. 12 Vahouny, G.V., Dennis, P., Chanderbhan, Noland, B.J. and Scallen, T.V. (1984) Biochem. Biophys. Res. Commun. 122. 509-515 R., Hinds, R.. Hodges, V.A. 13 Vahouny, G.V., Chanderbhan, and Treadwell, C.R. (1978) J. Lipid Res. 19. 570-577 110. 14 Tanaka, T. and Strauss, J.F., III. (1982) Endocrinology 1592-1598 15 Silber, R.H., Busch, R. and Oslapas, R. (1958) Clin. Chem. 4. 278-285 16 Londos. D. and Rodbell, M. (1975) J. Biol. Chem. 250. 3459-3465 J. and Saez, J.M. 17 Dazord, A., Morera, A.M., Bertrand, (1974) Endocrinology 95, 352-359 18 Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 19 Glynn, P., Cooper, D.M.F. and Schulster. D. (1977) Biothem. J. 168, 277-282

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