Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8310-8314, December 1985 Biochemistry Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21 (liposomes/adenylate cyclase/guanine nucleotide-binding protein/oncogene/transducin) SU-CHEN TSAI*, RONALD ADAMIK*, JOEL MOSS*, MARTHA VAUGHAN*, VEERASWAMY MANNEt, AND HSIANG-FU KUNGt *Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and tHoffmann-La Roche, Inc., Department of Molecular Oncology, Roche Research Center, Nutley, NJ 07110 Communicated by John J. Burns, July 29, 1985

ABSTRACT The Ha-ras protooncogene product p21, stimulatory or inhibitory receptors, respectively, to the which may be involved in control of cellular growth, is a regulation of adenylate cyclase (7, 19, 20). Gs and Gi are membrane protein that binds guanine nucleotides and hydro- active when GTP is bound to the a subunit; hydrolysis of lyzes GTP. p21 GTPase activity is stimulated by lysophospha- bound GTP to GDP and Pi results in inactivation (7, 19). tidylcholine; a delay in activation was observed unless p21 was Hormones and neurotransmitters acting through specific incubated with the phospholipid prior to assay. Maximal receptors enhance release of GDP from Gi or Gs, thereby, activation by the phospholipid was observed over a narrow accelerating the binding of GTP and activation of these concentration range; the presence in the assay mixture of guanine nucleotide-binding proteins (7, 19, 20). A similar lysophosphatidylcholine at concentrations above this optimum mechanism for signal transmission is utilized in the light- markedly inhibited p21 GTPase. GTP hydrolysis was also activated phosphodiesterase system of retina (21). The pho- stimulated, but to a lesser degree, by phosphatidylcholine. ton receptor rhodopsin, in the presence of light, activates Phosphatidylinositol and phosphatidylserine did not signifi- transducin by promoting the exchange of GDP for GTP (11). cantly enhance GTPase activity. The stimulatory effect of The a subunit oftransducin (Ta) with bound GTP activates a phospholipid was mimicked, in part, by nonionic detergents. cyclic GMP phosphodiesterase; activation is terminated by p21 may be related to other GTPases, the regulatory guanine GTP hydrolysis (11, 22, 23). nucleotide-binding G proteins of the hormone-sensitive ade- The adenylate cyclase system is the target of bacterial nylate cyclase complex and transducin of the retinal light- toxins that affect cells by altering their cAMP content. activated phosphodiesterase system. The G proteins and Choleragen (cholera toxin) and Escherichia coli heat-labile transducin are heterotrimers; the a subunits possess GTPase enterotoxin activate adenylate cyclase by catalyzing the activity and the Py subunit complex along with agonist- transfer of ADP-ribose from NAD to the a subunit of Gs receptor complex or light-activated rhodopsin enhance GTP (24-28); this decreases GTPase activity and prolongs the hydrolysis. p21 GTPase activity was slightly stimulated by lifetime of the activated state (29). Conversely, pertussis rhodopsin, but, in contrast to the GTPase activity of toxin ADP-ribosylates Gi, thereby, inactivating it and block- transducin, stimulation was not light-dependent. GTP hydrol- ing the action of inhibitory agonists on adenylate cyclase ysis was enhanced somewhat by (3y subunit complex in the (30-34). This modification of Gi decreases the rate of GTP absence, but not in the presence, of rhodopsin. Like the G hydrolysis by interfering with the G.-inhibitory receptor proteins and transducin, activity of p21 was altered by ADP- interaction and, thus, exchange of GTP for GDP (32, 33, ribosylation. Modification of p21 catalyzed by an NAD: 35-37). Choleragen and pertussis toxin also catalyze the arginine ADP-ribosyltransferase purified from turkey eryth- ADP-ribosylation of transducin and decrease its GTPase rocytes decreased both GTPase activity and guanine nucleotide activity (38-41). As reported here, ADP-ribosylation of p21 binding activity. by an NAD:arginine ADP-ribosyltransferase purified from turkey erythrocytes (42, 43) decreased its GTPase activity Ha-ras p21 is one of a family of oncogene products that are and ability to bind GTP. guanine nucleotide-binding proteins and catalyze the hydrol- ysis ofGTP to GDP and Pi; the GTPase activity ofthe protein MATERIALS AND METHODS appears to correlate inversely with its ability to transform Materials. [32P]NAD, [32P]GTP, and [8-3H]GTP were pur- cells (1-6). The product of the cellular protooncogene exhib- chased from New England Nuclear; electrophoresis supplies its significantly higher GTPase activity than the modified p21 from Bio-Rad; Mops (4-morpholinepropanesulfonic acid) and product ofthe viral gene (2, 4, 6). Although the GTP turnover Lubrol PX from Sigma. NAD:arginine ADP-ribosyltransfer- numbers of the proteins differ by greater than an order of ase was purified as described (43). magnitude, the cellular and viral products have a similar Preparation of Proteins. Normal Ha-ras p21 protein syn- capacity to bind guanine nucleotides (2, 5). thesized in E. coli was purified as described (4). Briefly, 10 The membrane-associated Ha-ras p21 shares some homol- g of E. coli cells were suspended in 25 ml of phosphate- ogy of amino acid sequence with several other guanine buffered saline (Pi/NaCl), pH 8.0/5 mM EDTA/25% nucleotide-binding proteins in animal cells (7-9), including (wt/vol) sucrose/1% Triton X-100/25 mg of lysozyme. The those involved in transmembrane signaling, which also hy- suspension was Vortex mixed and then was frozen and drolyze GTP (10-18). In the hormone-sensitive adenylate quickly thawed three times. DNase I (4 mg) was added to the cyclase system, two guanine nucleotide-binding proteins, lysate. After incubation at 0°C for 20 min, the lysate was termed Gs and Gi, couple the agonist occupancy of either centrifuged at 25,000 x g for 15 min. The pellet was washed four times with 25% (wt/vol) sucrose in 1% Triton X-100. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Gi, inhibitory guanine nucleotide-binding protein; GS, in accordance with 18 U.S.C. §1734 solely to indicate this fact. stimulatory guanine nucleotide-binding protein. 8310 Downloaded by guest on September 29, 2021 Biochemistry: Tsai et al. Proc. Natl. Acad. Sci. USA 82 (1985) 8311 washed pellet was then dissolved in Pi/NaCl containing 3.5 RESULTS M guanidineHCl and extensively dialyzed against 3.5 M guanidine'HCl. Any material that precipitated after dialysis The p21 protein encoded by the Ha-ras protooncogene and was removed by centrifugation at 15,000 x g for 10 min, and synthesized in E. coli, bound and hydrolyzed GTP as report- the clear supernatant containing p21 protein was stored at ed (3, 4, 45). The rate of GTP hydrolysis was constant for 2 -200C. These preparations are stable for months without any hr in the standard assay and was enhanced by lysophospha- significant loss of activity. tidylcholine (20 ,ug/ml) after a brief delay (Fig. 1). The delay Assay ofGTPase Activity. GTPase activity was measured as was abolished by incubation of p21 with lysophosphatidyl- described (4). Reaction mixtures contained p21 (usually 2 choline for 30 min at 37°C before assay (data not shown). ,pg), 350 mM guanidine, 0.2 mM MgCI2, bovine serum Maximal increases in activity (150-200%) were observed albumin 5 mM 2 over a very narrow concentration range (20-30 ,g of lyso- (2 jig), dithiothreitol, ,4M [y-32P]GTP [30 phosphatidylcholine/ml); at >60 pg of lysophosphatidylcho- Ci/mmol (6 x 101 cpm), 1 Ci = 37 GBq; New England line/ml, the GTPase was inhibited (Fig. 2). Phosphatidylcho- Nuclear], 20 mM Mops (pH 7.5), and other additions as line only slightly increased GTP hydrolysis whereas phos- indicated (total volume 100 1.L). After incubation for 2 hr at phatidylinositol was ineffective and phosphatidylserine was 370C (unless otherwise stated), 0.5 ml of charcoal suspension inhibitory (Table 1). Phosphatidylcholine (20 ,ug), which [12% (wt/vol) acid-washed Norite in 20 mM potassium increased activity only =20%, markedly decreased both the phosphate, pH 7.0] (14) was added, and the mixture was stimulatory and inhibitory effects oflysophosphatidylcholine centrifuged. A sample of supernatant (200 ,ul) was removed (Fig. 2). Nonionic detergents, Triton X-100 and Lubrol PX, for assay of 32p. Blank values (from assays containing all and the Zwitterionic detergent, CHAPS, 3-[(3-cholamidopro- additions except p21) were subtracted before calculation of pyl)dimethylammonio]-1-propanesulfonate, increased GTPase GTPase activity. Triplicate data points from representative activity (Table 2) '100%; sodium cholate, and the other de- experiments, which were repeated at least three times, are tergents, at high concentrations, inhibited GTPase activity reported and are presented with the SE. Phospholipids (1 (Table 2 and data not shown). Thus, it appears that like other mg/ml) were dispersed by sonication in 20 mM Hepes membrane proteins the effects of phospholipids on activity [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH were reproduced in part by nonionic detergents. 7.5, containing 1 mM EDTA and 1 mM dithiothreitol. The rate of GTP hydrolysis by the a subunits of transducin ADP-Ribosylation of p21 by Turkey Erythrocyte Transfer- or G0 was greatly enhanced by the presence oflight-activated ase. After dialysis against 1 M urea to remove guanidine, (photolyzed) rhodopsin incorporated in phosphatidylcholine samples of p21 (12 ,ug) were incubated at 25°C with 0.3 jig of liposomes and by the jry subunits of transducin (11, 14, 18, purified turkey erythrocyte ADP-ribosyltransferase in 20mM 23). p21-mediated GTP hydrolysis was greater in phosphati- Tris HCl, pH 7.5, containing 350 mM urea, 1 mM [32P]NAD dylcholine liposomes containing rhodopsin than in liposomes (1 without rhodopsin (Fig. 3). This difference was most notice- ,Ci), and 5% (vol/vol) propylene glycol (total volume 20 able in assays with lower amounts of p21. Maximal stimula- ,ul). Incubation was terminated by addition of 1 ml ofcold 5% tion of GTPase activity by either rhodopsin-phosphatidyl- (wt/vol) trichloroacetic acid and 10 ,ul of0.2% bovine serum choline vesicles or rhodopsin-free vesicles was observed albumin. Precipitated proteins were solubilized in 1% when low concentrations of p21 were present in the assay NaDodSO4 with 5% (vol/vol) 2-mercaptoethanol, 125 mM (Fig. 3 and Table 3); percentage stimulation by rhodopsin Tris base, 10% (vol/vol) glycerol and 0.0015% bromophenol decreased with amounts of p21 greater than those used in blue at 65°C for 10 min. Electrophoresis was carried out in most assays. The inclusion of rhodopsin also altered the 12% polyacrylamide gels containing 1% NaDodSO4 (44). effects of phospholipids on p21 GTPase activity. In the Gels were stained with Coomassie blue and dried. Dried gels presence of rhodopsin-containing liposomes, lysophospha- were exposed to Kodak XAR film for 1-2 hr. Protein was tidylcholine had no effect, although with liposomes alone (4.7 quantified by laser densitometry of stained gels. By compar- ,ug of phosphatidylcholine), increasing the concentration of ison with known amounts of albumin applied directly to the lysophosphatidylcholine first increased and then decreased gel, recovery of albumin from experimental samples was the GTPase activity (Fig. 4). The stimulatory effect of 60%. Estimated in a similar way recovery of p21 from rhodopsin on p21 GTPase activity differed from that in the unincubated mixtures was 55-60%. Gels were sliced (3.5 transducin system since light-activated and dark-adapted mm) for assay of 32P. By using total radioactivity in the area corresponding to ADP-ribosylated p21 and by assuming its recovery (which could not be estimated effectively by 8.0 densitometry) approximated that of p21 from unincubated m mixtures on the same gel, the ADP-ribose incorporated into E p21 was calculated and reported as mol ADP-ribose per mol p21. 5 6.0 [3H]GTP Binding Assay. Samples of p21 (12 ,g) in urea ._- were incubated with and without transferase exactly as described for [32P]ADP-ribosylation, except that 1 mM <, 4.0 unlabeled NAD was used. After 10 min at 25°C, 15 ,ul of 430 mM urea was added, and samples were taken for assay of ,(A 2.0 [3H]GTP binding (3). Reaction mixtures (total volume 50 ,ul) contained p21 (6.8 ,ug), 1 mM dithiothreitol, 5 mM MgCl2, bovine serum albumin (2 ;kg), 80 mM NaCl, 700 mM urea, 30 mM TrisHCl (pH 7.5), and [3H]GTP (4 ,uCi; 8.5 Ci/mmol). 30 60 90 120 After incubation for 60 min at 30°C, a sample (40 ,u) was Incubation, min transferred to a 25 mm 0.45-,m type HA filter (Millipore). FIG. 1. Effect of lysophosphatidylcholine on p21-mediated GTP Filters were quickly washed with 16 ml of ice cold 20 mM hydrolysis. GTPase activity ofp21 (2 ,ug) was assayed in the absence Tris HCl (pH 7.4), containing 100 mM NaCl, 1 mM dithio- (e) or in the presence (o) of lysophosphatidyicholine (2 Ag) for the threitol, and 5 mM MgCl2 and were dried before assay of 32p. indicated time. Downloaded by guest on September 29, 2021 8312 Biochemistry: Tsai et al. Proc. Natl. Acad. Sci. USA 82 (1985)

Table 2. Effects of detergents on p21-mediated GTP hydrolysis 6.00 GTPase activity, pmol per mg per 2 hr Detergent, Triton E 5.0 % Lubrol X-100 CHAPS Cholate 4.0 0.003 3680 ± 35 2880 ± 35 2530 ± 81 2300 ± 12 0.03 5410 ± 12 5870 ± 115 3110 ± 115 2760 ± 173 3.0 0.3 3910 ± 115 3570 ± 115 3450 ± 150 2650 + 150 Basal GTPase activity was 2852 ± 230 pmol per mg per 2 hr. U 2.0 [3H]GTP binding 54 ± 12% from 330 pmol/mg (n = 4). ; 1.0 Incubation with transferase for longer times (40-60 min) resulted in increased incorporation of ADP-ribose up to 4 1 2 3 4 S 6 7 mol per mol of p21 with no further decrease in [3H]GTP Lysophosphatidylcholine, ,ug binding or GTPase activity (Fig. 5). FIG. 2. Effect of lysophosphatidylcholine and phosphatidylcho- DISCUSSION line on p21-mediated GTP hydrolysis. GTPase activity of p21 (2 ,g) was assayed in the absence (o) or in the presence (e) of phosphati- Since p21 is membrane-associated in the cell, the effects of dylcholine (20 ,ig) and various concentrations of lysophos- phospholipids on its GTPase activity were investigated. phatidylcholine as indicated. Phosphatidylcholine, which forms multilamellar liposomes that can mimic a native membrane environment, increased rhodopsin were equally effective (Table 3). GTP hydrolysis activity slightly; phosphatidylserine and phosphatidylinositol was increased somewhat by transducin /3y subunits in the did not. Activation by lysophosphatidylcholine (maximally absence but not in the presence of rhodopsin (Table 3). =200%) occurred over a very narrow concentration range; For experiments with the NAD:arginine ADP-ribosyltrans- above the critical micellar concentration it was inhibitory. ferase, it was necessary first to remove guanidine (an alter- The stimulation could be in part due to a detergent action, and native ADP-ribose acceptor) from the p21 preparation. This in fact, nonionic detergents also increased GTP hydrolysis was accomplished by dialysis against 1 M urea. After incu- somewhat. bation for 10 min with the transferase and [32P]NAD, the The GTPase activity of the cellular protooncogene prod- ADP-ribosylated p21 migrated in a broad band on NaDod- uct, Ha-ras 21, assayed in the presence of lysophosphatidyl- S04/polyacrylamide gels more slowly than did the native choline was inhibited by arginine-specific ADP-ribosylation. protein (Fig. SA). Because ofits electrophoretic behavior, the The mechanisms ofthe effects ofADP-ribosylation by certain amount of modified p21 could not be determined by bacterial toxins on the GTPase activity of transducin have densitometry. Assuming that recovery was the same as that been defined. Inhibition of GTP hydrolysis by choleragen of p21 from the zero time sample (Fig. SA, lane 3), assay of results from decreased intrinsic GTPase activity of transdu- 32p in gel slices indicated incorporation of =3 mol of ADP- cin as revealed by single-turnover experiments (39). Pertussis ribose per mol of p21. Samples of p21 that were incubated toxin-catalyzed ADP-ribosylation decreases the ability of with or without transferase under the same conditions except transducin to interact with rhodopsin and to bind guanine that unlabeled NAD was used were assayed for GTPase nucleotide thereby decreasing GTP hydrolysis (40). The activity (with lysophosphatidylcholine, 2 ,g) and [3H]GTP effect on p21 of binding. ADP-ribosylation decreased GTPase activity 54 + ADP-ribosylation by the erythrocyte trans- 14% 2 ± = ferase appears similar to that produced by pertussis toxin from 1500 pmol per mg per hr (mean SE, n 6) and modification of transducin, i.e., inhibition of GTP hydrolysis resulted at least in part from inhibition of guanine nucleotide Table 1. Effects of phospholipids on p21-mediated GTP hydrolysis GTPase activity, 16 Additions, ,ug pmol per mg per 2 hr 14 None 1850 ± 140 6. L. 12 LysoPtdCho 2 6250 ± 289 10 PtdCho 2 1850 ± 90 Ua1) 8 10 1900 ± 38 -60. E 30 2350 ± 70 0._ 6 45 2450 ± 125 CZ 4 60 2400 ± 190 CL4 2 Ptdlns 2 2050 ± 82 7.5 2050 ± 155 15.0 1600 ± 322 1 2 3 4 5 6 Protein, ,ug PtdSer 2 1960 ± 201 7.5 1050 ± 240 FIG. 3. Effect of rhodopsin on p21-mediated GTP hydrolysis. The GTPase activity of various amounts of p21 protein was assayed 15.0 1400 ± 339 alone (o), or in the presence of phosphatidylcholine liposomes (4.7 GTPase activity of p21 (2 A.g) was assayed with the indicated ug) alone (e) or phosphatidylcholine liposomes containing rhodopsin additions. LysoPtdCho, lysophosphatidylcholine; PtdCho, phospha- (1.5,ug, A). Phosphatidylcholine liposomes with and without purified tidylcholine; PtdIns, phosphatidylinositol; PtdSer, phosphatidyl- rhodopsin were prepared as described (14). The assays were done in serine. triplicate, and the error bars indicate the SE. Downloaded by guest on September 29, 2021 Biochemistry: Tsai et A Proc. Natl. Acad. Sci. USA 82 (1985) 8313 Table 3. Effect of rhodopsin and transducin fry subunit complex A B on p21-mediated GTP hydrolysis GTPase activity, pmol per mg per 2 hr 94 kDa Addition(s) Light Dark 67 kDa None 940 ± 180 1000 ± 20 Rhodopsin (1.5 ,g) 3400 ± 80 3700 ± 370 45 kDa Transducin, Ary subunit complex (1 jig) 2000 ± 160 1400 ± 200 Rhodopsin and fry subunit complex (1 ug) 3300 ± 400 3100 ± 310 Si' "- 30 kDa GTPase activity of p2l (1.5 ;Lg) was assayed with phosphatidyl- choline (4.7 pLg), and the indicated additions in the light (as in other experiments) and in the dark. For this experiment, dark-adapted 20 kDa rhodopsin (14) was used. Transducin fry subunits were prepared as described (14). 1 2 3 4 1 2 3 FIG. 5. [32P]ADP-ribosylation of p21 catalyzed by purified binding. Of interest is the fact that multiple sites, probably NAD:arginine ADP-ribosyl transferase. Samples containing ADP- arginine residues, in p21 were modified by the transferase. ribosyl transferase (0.3 ,g), 1 mM [32P]NAD (1 uCi), 350 mM urea, The bacterial toxins rather selectively ADP-ribosylate a and 50 mM Tris-HCl (pH 7.5) with or without p21 (12 Mg) were single critical amino acid in transducin (46, 47). incubated for the indicated time at 250C. (A) Stained gel. Lanes: 1, The guanine nucleotide-binding proteins, and G, and standard proteins; 2, minus p21 incubated 10 min; 3, complete, zero Gi time; 4, complete, incubated 10 min. (B) Autoradiogram. Lanes: 1, are heterotrimers of a, (3, and y transducin composed minus p21 incubated 10 min; 2, complete, zero time; 3, complete, subunits (7, 19, 22, 48). The a subunits bind guanine nucle- incubated 10 min. otides, and are the targets of toxin-catalyzed ADP-ribosyl- ation (7, 19). The 8 and 'y subunits appear to be necessary for guanine nucleotide exchange and maximal expression of of these proteins and may be unrelated to a requirement for GTPase activity (14, 23). Transducin and the G proteins are interaction with common helper (e.g., /3'y subunit complex) coupled to receptors that affect the rate ofGTP hydrolysis (7, and receptor proteins. It has been proposed that the two ras 13-15, 18-20, 23, 49). In the presence of photolyzed proteins in yeast are, in fact, the guanine nucleotide-binding rhodopsin or agonist-occupied receptors guanine nucleotide components of adenylate cyclase (54-56). Alternatively, the exchange is enhanced, and the rate of GTP hydrolysis is fact that p21 has sequences that are homologous to both a and increased. Toxin-catalyzed ADP-ribosylation can interfere y subunits suggests that it is a hybrid of the transducin with intrinsic GTPase activity, nucleotide exchange, or complex, and, thus, perhaps helper-protein independent (9). coupling to receptor (29, 32, 38-41, 50). At present, proteins Several regulatory guanine nucleotide-binding proteins that serve as the equivalents of the ligand receptor and the appear to be in an active state when GTP or a nonhydrolyz- and 'y subunits of G proteins or transducin have not been able GTP analogue such as [S]pppG is bound (7). When GDP identified in the p21 system. The relatively low GTPase is bound, the protein is in an inactive form (57). Similarly, activity of p21 may reflect the absence of appropriate protooncogene product p21 is presumably active when GTP accessory but its GTPase was not is bound (p21 GTP); hydrolysis of GTP yields the inactive components, activity species, p21 GDP. The transformed protein binds GTP but enhanced by the (B-y subunit complex of transducin or by hydrolyzes it much more slowly. Although the role of p21 in rhodopsin in a light-dependent manner. The sequence ho- cell function has not been established, it is believed to be mology between p21 and the a subunits of transducin and involved in the regulation of growth (58). The growth pro- other guanine nucleotide-binding proteins (7, 8, 51-53) may moting activity of the transformed protein may be enhanced only reflect the nucleotide-binding and hydrolase properties as a result of its decreased GTPase activity, since this would prolong the lifetime of the active protein-GTP complex. As 77.0 reported here, ADP-ribosylation in vitro can alter the guanine nucleotide-binding properties of p21 and, therefore, the rate 66.0 at which it hydrolyzes GTP. In this regard, it resembles some other guanine nucleotide-binding regulatory proteins, and it E s-5.0- will be important to learn whether similar modification in the cell may be involved in the regulation of p21. O4.0 We thank Dr. Yasunori Kanaho for providing rhodopsin and Mrs. >~ 3.0 Barbara Mihalko for expert secretarial assistance. C5 1. Newbold, R. (1984) Nature (London) 310, 628-629. 2. Gibbs, J. B., Sigal, I. S., Poe, M. & Scolnick, E. M. (1984) C 2.0 Proc. Nad. Acad. Sci. USA 81, 5704-5708. 3. Manne, V., Yamazaki, S. & Kung, H.-F. (1984) Proc. Nad. Acad. Sci. USA 81, 6953-6957. 1 2 3 4 4. Manne, V., Bekesi, E. & Kung, H.-F. (1985) Proc. Nad. Acad. Lysophosphatidyicholine, Mg Sci. USA 82, 376-380. 5. McGrath, J. P., Capon, D. J., Goeddel, D. V. & Levinson, FIG. 4. Effect of lysophosphatidylcholine with and without A. D. (1984) Nature (London) 310, 644-649. rhodopsin on p21-mediated GTP hydrolysis. GTPase activity of p21 6. Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., was assayed with phosphatidylcholine-containing liposomes (4.7 Mg) Rosenberg, M. & Gross, M. (1984) Nature (London) 311, containing various amounts of lysophosphatidylcholine in the ab- 273-275. sence (o) or in the presence (A) of rhodopsin (1.5 Mg). 7. Gilman, A. G. (1984) Cell 36, 577-579. Downloaded by guest on September 29, 2021 8314 Biochemistry: Tsai et al. Proc. Natl. Acad. Sci. USA 82 (1985)

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