Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation

Transcription

1 Proc. NatL Acad. Sci. USA Vol. 79, pp , May 1982 Biochemistry Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein (rat C6 glioma cel/3-adrenergic receptor/nad/guanine nucleotide regulatory protein) TOSHIAKI KATADA AND MICHIO UI* Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Communicated by C. R. Park, February 17, 1982 ABSTRACT GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC ] in washed membranes prepared from C6 glioma cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, if the incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system. Islet-activating protein (lap) has recently been isolated from the culture medium of Bordetella pertussis (1, 2) as one of the pertussis toxins (3, 4). Injection of lap into animals in vivo (5) or addition of it to intact cell preparations in vitro (6-8) markedly modifies cellular camp responses to a variety of receptor agonists; receptor-mediated stimulation of camp accumulation in cells is potentiated, whereas receptor-mediated inhibition is abolished. This action of lap has been observed with rat pancreatic islet cells (5-7), rat cardiac cells (8), C6 glioma cells (9), 3T3 fibroblasts, and NG hybrid cells (unpublished data). The lap-induced modification of these camp responses seems to be due to a change in generation of camp via adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC ], rather than to its breakdown by phosphodiesterase, since it was observed in the presence ofan inhibitor ofthe diesterase. Indeed,,-adrenergic receptor-mediated increases of the membrane cyclase activity were enhanced and a-adrenergic receptor-mediated decreases were attenuated by previous treatment with lap of C6 glioma cells (9) and pancreatic islet cells (10), respectively. Thus, a mechanism whereby adenylate cyclase is linked to membrane receptors would be affected by lap. Exposure of intact cell preparations to lap has thus far been the sole means to achieve lap-induced modification of membrane adenylate cyclase responses, because addition of the pertussis toxin to cell-free preparations such as diluted homogenate or washed membranes was without effect. In this paper, we show that direct addition of lap to the membrane preparation from rat C6 glioma cells immediately enhanced GTP-dependent adenylate cyclase activity only in the presence of NAD and ATP. The enhancement was associated with covalent incorporation of radioactivity from the ADP-ribose moiety of NAD into a membrane protein with a Mr of 41,000. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C solely to indicate this fact. MATERIALS AND METHODS Materials. lap was purified by our colleagues (Research Laboratories, Kakenyaku Kako, Shiga, Japan) from a 3-day culture supernatant ofb. pertussis (Tohama strain, phase I) as described (1). lap was dissolved (1 mg/ml) in a vehicle of 0.1 M potassium phosphate buffer, ph 7.0/2 M urea; the solution was stored at 40C until use. The vehicle was used instead of the lap solution as a control. [a-32p]nad (25 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels), nicotinamide [U-14C]adenine dinucleotide (286 mci/mmol), [carbonyl-14c]nad (53 mci/mmol), and [14C]- methylated protein mixture (CFA-626) were purchased from Amersham. [ribose-u-14c]nicotinamide adenine dinucleotide was a gift from 0. Hayaishi, Kyoto. NAD, ATP, GTP, and i-isoproterenol were from Sigma, and snake venom phosphodiesterase was from Worthington. Other materials were from sources described (5-10). Anti-lap serum prepared by immunizing rabbits with lap as described (6) was fractionated to provide the gamma-globulin fraction, which was used as "anti-iap antibody" in an amount sufficient to neutralize lap in the reaction mixture. C6 Glioma Cell Culture and Preparations of Crude Membranes and Cell Sap. Methods for C6 glioma cell culture and the total homogenate or crude membrane preparation therefrom have been described (9). These preparations were made in 25 mm Tris.HCI/2.5 mm MgCl2, ph 7.5 (membrane buffer), at a final concentration of 5-10 mg of protein/ml and stored in liquid nitrogen until use. Cell sap was obtained by lysis of the packed cells in an equal volume of membrane buffer, followed by homogenization and centrifugation at 100,000 x gfor30 min. Where indicated, it was mixed with 1/10 vol of Norit A and stored at 0 C for 30 min before centrifugation to give "charcoaltreated cell sap," which is depleted ofendogenous nucleotides. Protein was determined by the method of Lowry et al (11) using bovine serum albumin as standard. Treatment of Membranes with lap and their Radiolabeling. The crude membrane preparation or the total homogenate of C6 glioma cells (100,ul) was incubated with lap or cholera toxin for 5 min (unless otherwise stated) at 37 C. Further additions are indicated in the figures and Table 1. For radiolabeling, the reaction mixture was supplemented with [a-32p]nad (10,uM; 5 Ci/mmol, unless otherwise specified) or another labeled NAD, 1 mm ATP, and 10 mm thymidine, and incubation was prolonged up to 10 min. At the end of incubation, the reaction mixture was quickly diluted with 1 ml of ice-cold membrane buffer and then centrifuged at 10,000 x g for 3 min; the sediment. was washed twice by suspension and centrifugation to provide lap-treated or radiolabeled membrane preparation Abbreviation: lap, islet-activating protein. * To whom reprint requests should be addressed.

2 3130 Biochemistry: Katada and Ui Table 1. NAD and ATP requirements for Iap enhancement of GTP activation of membrane adenylate cyclase Adenylate cyclase activity, pmol of camp per mg of protein per min Addition Without Iap (a) With Iap (b) b/a None Cell sap* Heated cell sapt Charcoal-treated cell sap mm NAD mm ATP mm NAD/1 mm ATP mm NAD/1 mm ATP mm nicotinamide The membrane fraction (200 Ag of protein) was incubated for 5 min with or without Iap at 10 j.g/ml in the presence of the additions indicated. After incubation, membranes separated from the reaction mixture were washed and assayed for adenylate cyclase in thepresence of 10 JAM GTP. * Final concentration, 8 mg of protein/ml. t Cell sap was heated for 1 min at 90 C before use. Adenylate Cyclase Assay. This was conducted essentially as described (9, 10). Washed membranes (20-30,ug of protein) were incubated for 5 min at 37 C in 100,1 of 50 mm Tris-HCl/ 0.5 mm ATP/5 mm phosphocreatine/5 mm MgCl2/0.5 mm 3-isobutyl-1-methylxanthine/1 mm EGTA, ph 7.5, containing creatine kinase at 50 units/ml and bovine serum albumin at 1 mg/ml. Further additions are shown in the figures and Table 1. Assays were carried out in duplicate, and cyclase activities are given as mean values in picomoles ofcamp synthesized per mg of protein per min. A sensitive and specific radioimmunoassay (12) was used to determine camp generated. Gel Electrophoresis and Autoradiography. Radiolabeled membranes were dissolved in gelsample buffer (1% NaDodSOJ 5% 2-mercaptoethanol/10% glycerol/62.5 mm Tris'HCl/0.02% bromophenol blue, ph 6.8) and heated for 3 min at 100 C. An aliquot (=50,ug of protein) was loaded on each lane of a 1-mmthick polyacrylamide slab gel (main gel, 12.5% acrylamide; stacking gel, 4.5% acrylamide/0. 1% NaDodSO4) and subjected to electrophoresis as described by Laemmli (13). After electrophoresis, the gels were stained with Coomassie brilliant blue R-250, destained, and dried on Whatman 3MM filter paper. The dried gel was autoradiographed at -80 C using Kodak X- Omat film. The Mr markers were [I4C]methylated proteins: myosin (Mr, 200,000), phosphorylase b (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lysozyme (14,300). RESULTS Direct Action of lap on Adenylate Cyclase of Broken-Cell Preparations from C6 Glioma Cells. Since our preliminary experiments showed that direct addition of lap to washed membrane preparations from various cells including C6 glioma failed to affect the membrane adenylate cyclase, we used a concentrated homogenate of C6 cells, instead of washed membranes, in the experiments shown in Fig. 1. When the condensed homogenate was incubated with lap for 5 min, there were marked changes in the activity of adenylate cyclase of the membrane prepared therefrom; increasing the lap concentration in-the incubation medium from 80 to 2,000 ng/ml increased the adenylate cyclase activity estimated in the presence of GTP (Fig. 1A). Isoproterenol-induced stimulation of the cyclase was also enhanced by Iap, though much less than was GTP stimulation. t. fi 4) bl tn co 0, I<'I '0 IE wi' Proc. Natl. Acad. Sci. USA 79 (1982) * I. I , IAP, ng/ml Homogenate, mg/ml FIG. 1. Adenylate cyclase activity of cell homogenate incubated with lap. The total homogenate fraction of C6 glioma cells was incubated with lap for 5 min. Then, the reaction mixture was diluted and centzifuged to obtain the crude membrane fraction and that fraction was washed twice and assayed for adenylate cyclase activity without any additions (o) or with 10 JM isoproterenol (A), 10/M GTP (o), 10 mm NaF (.), or GTP/isoproterenol (A). (A) The homogenate at 20 mg of protein/ml was incubated with Iap at various concentrations. (B) The homogenate at various concentrations was incubated with lap at 2 pg/ml. The action of NaF, however, was not affected by lap, nor was the basal activity estimated in the absence of these effectors. These modifications of membrane adenylate cyclase caused by direct exposure of the broken-cell preparation to lap were the same as those previously observed with membranes prepared from lap-treated intact cells (9). The more concentrated the homogenate used, the stronger was the lap action (Fig. 1B), suggesting that factor(s) present in the cell sap were required for lap action. When the washed membrane preparation was used instead of the concentrated homogenate, lap was without effect on membrane adenylate cyclase activity subsequently assayed with GTP unless ATP was included in the lap treatmcnt medium (Fig. 2). Thus, ATP is one of the factors essential for lap action. Addition of the cell sap in increasing amounts enhanced lap activation of GTP-dependent adenylate cyclase activity progressively either with or without ATP, indicating that factor(s) other than ATP are also responsible for lap action. NAD and ATP Requirement for the Direct Action of lap on Membrane Adenylate Cyclase. The action of the cell sap to support Iap-induced activation of GTP-dependent adenylate cyclase activity was observed even with sap that had been heated but not with sap that had been depleted of endogenous nucleotides by charcoal treatment (Table 1). NAD appeared to be particularly important, since addition of NAD to the charcoal-treated cell sap was effective in restoring lap action. This action of NAD was not mimicked by other analogs or breakdown products of NAD including adenine or guanine nucleotides, nicotinamide, or ADP-ribose (data not shown). The reduced form of NAD, NADH, or NADP could replace NAD to a limited extent (data not shown). Even at a high NAD concentration, addition of ATP enhanced lap action. Probably, membrane adenylate cyclase is gradually inactivated unless the medium is fortified with ATP, as suggested by the fact that the basal cyclase activity of membranes is lower in the absence ofatp than in its presence (Table 1 and Fig. 2). Conversely, lap action supported by NAD and ATP was suppressed by a high concentration of nicotinamide (Table 1) but not by other breakdown products of NAD, such as AMP, ADP, or ADP-ribose (data not shown).

3 > >. _ C; b.d m -To W a Biochemistry: Cell sap, mg/ml Katada and Ui Iap, ug/ml FIG. 2. NAD and ATP requirements for Iap enhancement of GTP activation of particulate adenylate cyclase. (A) Mixtures of crude membrane fractions (200 pg of protein) and various concentrations of cell sap were incubated for 5 min without (o and A) or with (e and A) Iap at 10 jg/ml in the presence (A and *) or absence (o and ) of 1 mm ATP. (B) The crude membrane preparation (200 ug of protein) was incubated with various concentrations of Iap in the absence (o) or presence of 1 mm NAD (e), 1 mm ATP (v&, or NAD plus ATP (n). In both A andb, the particulate was separated from the reaction mixture, washed, and assayed for adenylate cyclase activity with 10 jim GTP. When the washed membranes were incubated with lap in the presence of both NAD and ATP, the minimum concentration of lap to enhance GTP activation of adenylate cyclase activity was. 05 Ag/ml and increases in the concentration of lap increased the enhancement progressively (Fig. 2B). lap was still slightly effective on omission of either ATP or NAD, probably because of contamination of washed membranes with endogenous nucleotides. Time Course of lap Action on Membrane Adenylate Cyclase. Washed membranes were incubated with lap in the presence of NAD and ATP for periods of 2-10 min and then analyzed for adenylate cyclase activity in the presence ofgtp. The cyclase activity is plotted as a function of time of lap treatment in Fig. 3. GTP-stimulated adenylate cyclase activity increased immediately after addition of lap. The increase was initially linear and the rate of this initial increase (the slope of plots at 0 time in Fig. 3) was proportional to the concentration of lap from 1 to 25 llg/ml. When the anti-iap antibody was added at 4 min of lap (5 Ag/ml) treatment, there was no further increase in cyclase activity nor any decrease in activity. Thus, lap appears to catalyze irreversible modification of membrane adenylate cyclase and progressively render the enzyme more susceptible to GTP (and (3-adrenergic agonists). At the highest concentration (25 Ag/ml) of lap, the increase in adenylate cyclase activity tended to level off within 10 min, probably reflecting saturability of the lap modification. lap-catalyzed ADP-ribosylation of the Membrane Protein ofc6 Glioma Cells. The strict NAD requirement, together with the nicotinamide-induced reversal, of the lap action suggests that NAD is one ofthe substrates and nicotinamide is one ofthe products ofthe Iap-catalyzed reaction. lap treatment ofwashed membranes was hence carried out with [a-32p]nad. The 32p content of the membrane protein fractions was then analyzed by NaDodSO4polyacrylamide gel electrophoresis. As shown in Fig. 4A, a protein with a Mr of 41,000 was predominantly labeled only when lap was present in the reaction mixture. Two minor radioactive bands (Mr, 36,000 and 29,000) were also observed in the presence of lap. When lap was replaced by cholera toxin in the incubation mixture, proteins with Mr values of A> E C) b.d 150 ax _) _ 100 [ >s - To Lo-0 Ao Preincubation time, min FIG. 3. Time course of enhancement of GTP-dependent.adenylate cyclase activity by various concentrations of lap. The crude membrane preparation (2 mg of protein) was incubated with lap in 1 ml of membrane buffer/i mm ATP/1 mm NAD. At various times, 100 pl of medium was withdrawn and the particulate fraction was prepared and assayed for adenylate cyclase activity in the presence of 10 pm GTP. lap concentration added at time 0: o, none; 9, 1 A, qg/ml; 5 pg/ml; *, 25 pg/ml. Anti-lap antibody was added at 4 min to the mixture containing lap at 5 ug/ml ( and A). 45,000 and 48,000/49,000 (doublet) were slightly labeled. Incubation of membranes with both lap and cholera toxin caused labeling of all of the protein bands labeled with either agent alone, indicating that the proteins labeled by lap are distinct A B C W -A 460 -D ^s -~~~E30-w I4.3* FIG. 4. NaDodSO4polyacrylamide gel electrophoresis of crude cell membranes incubated with radiolabeled NAD. (A) Membranes were incubated with Iap at 25 ug/ml (lanes 2 and 5) or preactivated cholera toxin at 25 ug/ml (lanes 4 and 5) in the presence of [a- 32P]NAD. Controls: lane 1, vehicle was added instead of toxin; lane 3, vehicle/200 um GTP was used. Cholera toxin was preactivated as described (21). The gel was exposed to x-ray film for 24 hr. A-: A, Mr 48,000/49,000 (doublet); B, Mr 45,000; C, Mr 41,000; D M 36,000; E, Mr 29,000. Lane 6: '4C-Labeled marker proteins (Mr 10-). (B).Mem- X Proc. Natl. Acad. Sci. USA 79 (1982) 3131 branes were incubated with Iap at 25 tug/ml and 125 pm (50 mci/ mmol) [carbonyl-14c]nad (lane 1), [ribose-u-'4c]nicotinamide adenine dinucleotide (lane 2), and nicotinamide [U-'4C]adenine dinucleotide (lane 3). The gel was exposed to x-ray film for 7 days. -A, Mr 41,000 band. (C) After incubation with [a-32p]nad and Iap at 25 plg/mi, the particulate was separated from the reaction mixture, washed, and further incubated for 2 hr at 37TC with (lane 2).or without (lane 1) snake venom phosphodiesterase at 30 units/ml (14). The gel was exposed to x-ray film for 20 hr.

4 3132 Biochemistry: Katada and Ui from the substrates of the similar cholera toxin-catalyzed reaction. lap-induced radiolabeling of the Mr 41,000 protein was also observed with nicotinamide [U-14C]adenine dinucleotide and with [ribose-u-'4c]nicotinamide adenine dinucleotide but was not with [carbonyl-'4cinad (Fig. 4B). When membrane labeled with [a-32p]nad in the presence of lap was incubated with snake venom phosphodiesterase, the Mr 41,000 protein lost most ofits radioactivity (Fig. 4C). Phosphodiesterase digestion is known to release 5'-AMP from ADP-ribosylated proteins (14). Incorporation of radioactivity into the Mr 41,000 protein was suppressed by unlabeled NAD or nicotinamide at a high concentration but not by unlabeled AMP, ADP, or ADP-ribose (Fig. 5A). These results suggest that lap catalyzes transfer ofthe entire ADP-ribose moiety of NAD to the Mr 41,000 protein of the C6 cell membrane. lap-induced 32p labeling was enhanced by addition of ATP (Fig. 5A), which was an essential factor for the lap enhancement of GTP-dependent adenylate cyclase activity (Fig. 2). The labeling increased as the concentration of lap was increased from 0.2 to 25 pig/ml (Fig. 5B). Addition of the anti-lap antibody during incubation with [a-32p]nad prevented further labeling of the Mr 41,000 protein. These characteristics of lap-induced 3P labeling are similar to those observed in Figs. 2 and 3 and Table 1 for lap-induced enhancement of GTP-dependent adenylate cyclase activity. Thus, ADP-ribosylation of the Mr 41,000 protein caused by lap appears to be responsible for its ability to enhance adenylate cyclase responses. DISCUSSION We have recently reported that GTP activation of adenylate cyclase in the membrane preparation from C6 cells is enhanced by exposure of intact cells to low concentrations of lap during culture (9). The action of lap probably results from its interaction with the guanine nucleotide regulatory protein in the membrane adenylate cyclase system. This enhancement of GTP activation was used, in the present study, as a measure of direct action of lap on the adenylate cyclase system of washed B iqa N OWN 4P FIG. 5. Radiolabeling of the Mr 41,000 protein by lap. The crude membrane fraction was labeled with (a-32p]nad and then subjected to NaDodSO4/polyacrylamide gel electrophoresis as described in Materials and Methods. The band of the Mr 41,000 protein on an autoradiogram is shown. (A) Labeling was carried out without lap (lane 1) or with lap at 25,g/ml (lanes 2-7). Additions: 1 mm ATP (lane 1), none (lane 2), ATP (lane 3), ATP/1 mm AMP (lane 4), ATP/1 mm ADP (lane 5), ATP/1 mm ADP-ribose (lane 6), ATP/50 mm nicotinamide (lane 7). (B) Labeling with 1 mm ATP and various concentrations of lap. Lanes: 1, 0.2 pyg/ml; 2, 1 jg/ml; 3, 5 pug/ml; 4, 25 pzg/ml; 5, anti-lap antibody was added 4 min after addition of lap at 5 Ig/ml. Autoradiogram was obtained after 36-hr exposure to x-ray film. Proc. Nad Acad. Sci. USA 79 (1982) cell membranes. It is shown here that the same enhancement was observed in cell membranes incubated with lap for a short time when the incubation mixture was supplemented with the concentrated soluble fraction of the cells. The soluble factors supporting the lap action were identified as NAD and ATP. The direct action of lap on washed membranes displayed apparent disparity from its action onintact cells in the following two respects. The first is the lag period so far observed on treatment of intact cells with lap. There was no lag in the direct action of lap on the membrane. Our detailed kinetic studies of the gradually developing action of lap to reverse a-adrenergic inhibition of insulin secretion from islet cells (6) showed that the delayed onset of lap action represents a true lag period rather than a slowly reacting process. It was concluded tentatively (6) that this lag period reflected the time for gradual insertion of lap molecules into the cell membrane before reaching the site oftheir action on the intracellular membrane surface. The present results thus support this conclusion by showing that the action of lap started immediately on direct contact with membranes. It is worthy of note in this regard that the time course of the development of lap action on membranes (Fig. 3) was similar to the time course for its action on intact cells that starts at the end of a lag period (6). The second discrepancy is the concentration of lap required. When intact C6 cells were incubated with lap for 6 hr. the concentration of lap to enhance GTP activation of adenylate cyclase of the membranes prepared therefrom was ng/ml (9). In contrast, ,000 ng/ml of lap was required for direct enhancement of the membrane GTP-dependent cyclase (Figs. 2 and 3). It should be emphasized, however, that the concentration of lap required was strictly dependent on the time of incubation in both intact cells and washed membranes. The concentration of lap to cause the half-maximal effect on intact C6 cells was 100, 1, and0.001 ng/ml for incubation times of 2, 6, and 18 hr, respectively (9), and for intact islet cells, it was 50, 3, and 0.1 ng/ml for 6, 12, and 24 hr of incubation (6). Thus, it is likely that, if the time of incubation of washed membranes could be prolonged to 1 to 2 hr, lap at a few nanograms per milliliter would directly enhance GTP activation of membrane adenylate cyclase toextents similar to those observed with intact cells incubated under the same conditions. Unfortunately, membrane adenylate cyclase of broken cell preparations was so fragile, even in the presence of ATP, that prolongation of incubation beyond 20. min caused rapid inactivation of the enzyme and thereby obscured its susceptibility to lap. Probably, discrepancies are only apparent. Incorporation of radioactivity from NAD into a membrane protein was enhanced by lap when the radioactivity was located in the ADP-ribose moiety, but no radioactivity was incorporated from the nicotinamide region. Protein that had been labeled with [a-32p]nad liberated 32P on hydrolysis with snake venom phosphodiesterase, indicating the presence of PP, bonding in the labeled protein molecule. Nicotinamide, another roduct ofadp-ribosylation, was very effective in inhibiting [a- PINAD incorporation. Thus, lap transfers the ADP-ribose moiety of NAD to a membrane protein. It is well known that -the other two kinds ofprotein of bacterial origin, diphtheria (15) and cholera (16) toxins; also catalyze ADP-ribosylation of GTP-binding proteins. The concentration dependence, ATP requirement, and susceptibility to nicotinamide inhibition of lap-induced incorporation of [a-32p]nad into the Mr 41,000 protein were the same as those of NAD-dependent lap enhancement ofgtp activation of membrane adenylate cyclase. Thus, lap possesses a catalytic action for (or induces) ADP-ribosylation ofa membrane protein, which could be responsible for its enhancement of GTP acti-

5 Biochemistry: Katada and Ui vation (and receptor coupling) ofthe membrane cyclase in intact cells (9) or washed membranes. The time dependence of this lap action may reflect this catalytic property of the pertussis protein. Moreover, duration of lap-induced enhancement of GTP-dependent adenylate cyclase activity even after addition of the anti-lap antibody (Fig. 3) is consistent with irreversible ADP-ribosylation of the membrane protein. Cholera toxin catalyzes ADP-ribosylation of one of the subunits of the guanine nucleotide regulatory component of the membrane adenylate cyclase system (17-20). Indeed, the Mr 45,000 and 48,000/49,000 proteins in C6 cell membranes were labeled with [a-32p]nad in the presence of preactivated cholera toxin (Fig. 4A). This cholera toxin-catalyzed labeling was strictly dependent on GTP and increased on addition ofchicken erythrocyte cytosol (unpublished), in accord with previous reports (18, 21). Although this GTP requirement was not observed with lap, it is possible that ATP, an essential factor for lap action, was effective in yielding GTP via phosphorylation of endogenous GDP. It is unlikely that the proteins ADP-ribosylated in the presence of lap are proteolytic products of cholera toxinspecific substrates, since the labeling of its own substrates by either toxin was not affected by simultaneous addition of the other (Fig. 4A). It is possible that the two toxins may cause ADP-ribosylation of different subunits of the guanine nucleotide regulatory protein. We thank Professor Osamu Hayaishi, Kyoto University Faculty of Medicine, for his generous gift of [ribose-u-_4c]nicotinamide adenine dinucleotide and valuable discussions. We also thank Professor Charles R. Park, Vanderbilt University School of Medicine, for his valuable advice in preparing the manuscript. This work was supported by research grants from the Scientific Research Fund of Ministry of Education, Science and Culture, Japan. Proc. Natl. Acad. Sci. USA 79 (1982) Yajima, M., Hosoda, K., Kanbayashi, Y., Nakamura, T., Nogimori, K., Nakase, Y. & Ui, M. (1978) J. Biochem. (Tokyo) 83, Yajima, M., Hosoda, K., Kanbayashi, Y., Nakamura, T., Takahashi, I. & Ui, M. (1978) J. Biochem. (Tokyo) 83, Ui, M., Katada, T. & Yajima, M. (1978) in International Symposium on Pertussis, eds. Manclark, C. R. & Hill, J. C. (National Institutes of Health, Bethesda, MD), publ. no , pp Munoz, J. J. & Bergman, R. K. (1978) in International Symposium on Pertussis, eds. Manclark, C. R. & Hill, J. C. (National Institutes of Health, Bethesda, MD), publ. no , pp Katada, T. & Ui, M. (1979)J. Biol Chem. 254, Katada, T. & Ui, M. (1980) J. Biol Chem. 255, Katada, T. & Ui, M. (1981) J. Biochem. (Tokyo) 89, Hazeki, 0. & Ui, M. (1981) J. Biol Chem. 256, Katada, T., Amano, T. & Ui, M. (1982) J. Biol Chem. 257, in press. 10. Katada, T. & Ui, M. (1981) J. Biol Chem. 256, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) 1. Biol Chem. 193, Honma, M., Satoh, T., Takezawa, J. & Ui, M. (1977) Biochem. Med. 18, Laemmli, U. K. (1970) Nature (London) 227, Lehmann, A. R., Kirk-Bell, S., Shall, S. & Whish, W. J. D. (1974) Exp. Cell Res. 83, Hayaishi, 0. & Ueda, K. (1977) Annu. Rev. Biochem. 46, Moss, J. & Vaughan, M. (1979) Annu. Rev. Biochem. 48, Cassel, D. & Pfeuffer, T. (1978) Proc. Natl Acad. Sci. USA 75, Gill, D. M. & Meren, R. (1978) Proc. Natl Acad. Sci. USA 75, Johnson, G. L., Kaslow, H. R. & Bourne, H. R. (1978) J. Biol Chem. 253, Northup, J. K., Sterweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (1980) Proc. NatL Acad. Sci. USA 77, Enomoto, K. & Gill, G. M. (1980)J. Biol Chem. 255,