Proc. NatL Acad. Sci. USAVol. 79, pp. 3129-3133, May 1982Biochemistry

Direct modification of the membrane adenylate cyclase systemby islet-activating protein due to ADP-ribosylationof 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 adenylatecyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] inwashed membranes prepared from C6 glioma cells was enhancedby incubation with islet-activating protein, one of the pertussistoxins, if the incubation mixture was supplemented with NAD andATP. The action of the protein was observed immediately afterits addition and increased progressively in magnitude as the pro-tein concentration or the incubation time increased. There wassimultaneous incorporation of radioactivity from the ADP-ribosemoiety of variously labeled NAD into the membrane protein witha molecular weight of 41,000. We conclude that islet-activatingprotein enhances receptor-mediated GTP-induced activation ofmembrane adenylate cyclase as a result of ADP-ribosylation of amembrane protein, probably one of the components of the recep-tor-adenylate cyclase system.

Islet-activating protein (lap) has recently been isolated from theculture medium of Bordetella pertussis (1, 2) as one of the per-tussis toxins (3, 4). Injection of lap into animals in vivo (5) oraddition of it to intact cell preparations in vitro (6-8) markedlymodifies cellular cAMP responses to a variety of receptor ag-onists; receptor-mediated stimulation of cAMP accumulationin cells is potentiated, whereas receptor-mediated inhibition isabolished. This action of lap has been observed with rat pan-creatic islet cells (5-7), rat cardiac cells (8), C6 glioma cells (9),3T3 fibroblasts, and NG 108-15 hybrid cells (unpublished data).The lap-induced modification of these cAMP responses seemsto be due to a change in generation ofcAMP via adenylate cy-clase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], ratherthan to its breakdown by phosphodiesterase, since it was ob-served in the presence ofan inhibitor ofthe diesterase. Indeed,,-adrenergic receptor-mediated increases of the membranecyclase activity were enhanced and a-adrenergic receptor-me-diated decreases were attenuated by previous treatment withlap ofC6 glioma cells (9) and pancreatic islet cells (10), respec-tively. Thus, a mechanism whereby adenylate cyclase is linkedto membrane receptors would be affected by lap.

Exposure of intact cell preparations to lap has thus far beenthe sole means to achieve lap-induced modification of mem-brane adenylate cyclase responses, because addition of the per-tussis toxin to cell-free preparations such as diluted homogenateor washed membranes was without effect. In this paper, weshow that direct addition of lap to the membrane preparationfrom rat C6 glioma cells immediately enhanced GTP-dependentadenylate cyclase activity only in the presence of NAD andATP. The enhancement was associated with covalent incorpo-ration of radioactivity from the ADP-ribose moiety of NAD intoa membrane protein with a Mr of 41,000.

MATERIALS AND METHODS

Materials. lap was purified by our colleagues (Research Lab-oratories, Kakenyaku Kako, Shiga, Japan) from a 3-day culturesupernatant ofB. pertussis (Tohama strain, phase I) as described(1). lap was dissolved (1 mg/ml) in a vehicle of 0.1 M potassiumphosphate buffer, pH 7.0/2 M urea; the solution was stored at40C until use. The vehicle was used instead of the lap solutionas a control. [a-32P]NAD (25 Ci/mmol; 1 Ci = 3.7 X 1010becquerels), nicotinamide [U-14C]adenine dinucleotide (286mCi/mmol), [carbonyl-14C]NAD (53 mCi/mmol), and [14C]-methylated protein mixture (CFA-626) were purchased fromAmersham. [ribose-U-14C]Nicotinamide adenine dinucleotidewas a gift from 0. Hayaishi, Kyoto. NAD, ATP, GTP, and i-iso-proterenol were from Sigma, and snake venom phosphodies-terase was from Worthington. Other materials were fromsources described (5-10). Anti-lap serum prepared by immu-nizing rabbits with lap as described (6) was fractionated to pro-vide the gamma-globulin fraction, which was used as "anti-IAPantibody" in an amount sufficient to neutralize lap in the re-action mixture.C6 Glioma Cell Culture and Preparations of Crude Mem-

branes and Cell Sap. Methods for C6 glioma cell culture andthe total homogenate or crude membrane preparation there-from have been described (9). These preparations were madein 25mM Tris.HCI/2.5mM MgCl2, pH 7.5 (membrane buffer),at a final concentration of 5-10 mg of protein/ml and stored inliquid nitrogen until use. Cell sap was obtained by lysis of thepacked cells in an equal volume of membrane buffer, followedby homogenization and centrifugation at 100,000 x gfor30 min.Where indicated, it was mixed with 1/10 vol of Norit A andstored at 0°C for 30 min before centrifugation to give "charcoal-treated cell sap," which is depleted ofendogenous nucleotides.Protein was determined by the method of Lowry et aL (11) usingbovine serum albumin as standard.

Treatment of Membranes with lap and their Radiolabeling.The crude membrane preparation or the total homogenate ofC6 glioma cells (100 ,ul) was incubated with lap or cholera toxinfor 5 min (unless otherwise stated) at 37°C. Further additionsare indicated in the figures and Table 1. For radiolabeling, thereaction mixture was supplemented with [a-32P]NAD (10 ,uM;5 Ci/mmol, unless otherwise specified) or another labeledNAD, 1 mM ATP, and 10 mM thymidine, and incubation wasprolonged up to 10 min. At the end of incubation, the reactionmixture was quickly diluted with 1 ml of ice-cold membranebuffer and then centrifuged at 10,000 x g for 3 min; the sedi-ment. was washed twice by suspension and centrifugation toprovide lap-treated or radiolabeled membrane preparation.

Abbreviation: lap, islet-activating protein.* To whom reprint requests should be addressed.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Table 1. NAD and ATP requirements for Iap enhancement ofGTP activation of membrane adenylate cyclase

Adenylate cyclase activity, pmol ofcAMP per mg of protein per min

Addition Without Iap (a) With Iap (b) b/a

None 28.8 31.3 1.09Cell sap* 21.1 93.7 4.44Heated cell sapt 24.9 89.7 3.60Charcoal-treated cell sap 26.7 32.5 1.22

1 mM NAD 21.3 60.6 2.851 mM ATP 37.4 47.6 1.271 mM NAD/1 mMATP 33.8 102.7 3.04

1 mM NAD/1 mM ATP 34.4 109.2 3.1750 mM nicotinamide 36.1 54.6 1.51

The membrane fraction (200 Ag of protein) was incubated for 5 minwith or without Iap at 10 j.g/ml in the presence of the additions in-dicated. After incubation, membranes separated from the reactionmixture were washed and assayed for adenylate cyclase inthepresenceof 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 asdescribed (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 mM3-isobutyl-1-methylxanthine/1 mM EGTA, pH 7.5, containingcreatine kinase at 50 units/ml and bovine serum albumin at 1mg/ml. Further additions are shown in the figures and Table1. Assays were carried out in duplicate, and cyclase activitiesare given as mean values in picomoles ofcAMP synthesized permg of protein per min. A sensitive and specific radioimmu-noassay (12) was used to determine cAMP generated.

Gel Electrophoresis and Autoradiography. Radiolabeledmembranes were dissolved in gel sample buffer(1% NaDodSOJ5% 2-mercaptoethanol/10% glycerol/62.5 mM Tris'HCl/0.02%bromophenol blue, pH 6.8) and heated for 3 min at 100°C. Analiquot (=50 ,ug of protein) was loaded on each lane of a 1-mm-thick polyacrylamide slab gel (main gel, 12.5% acrylamide;stacking gel, 4.5% acrylamide/0. 1% NaDodSO4) and subjectedto electrophoresis as described by Laemmli (13). After electro-phoresis, the gels were stained with Coomassie brilliant blueR-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 serumalbumin (69,000), ovalbumin (46,000), carbonic anhydrase(30,000), and lysozyme (14,300).

RESULTSDirect Action of lap on Adenylate Cyclase of Broken-Cell

Preparations from C6 Glioma Cells. Since our preliminary ex-periments showed that direct addition of lap to washed mem-brane preparations from various cells including C6 glioma failedto affect the membrane adenylate cyclase, we used a concen-trated homogenate of C6 cells, instead of washed membranes,in the experiments shown in Fig. 1. When the condensed ho-mogenate was incubated with lap for 5 min, there were markedchanges in the activity of adenylate cyclase of the membraneprepared therefrom; increasing the lap concentration in-the in-cubation medium from 80 to 2,000 ng/ml increased the ade-nylate cyclase activity estimated in the presence of GTP (Fig.1A). Isoproterenol-induced stimulation of the cyclase was alsoenhanced by Iap, though much less than was GTP stimulation.

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IE wI' * I . I0 80 400 2,000 3 6 12 24

IAP, ng/ml Homogenate, mg/ml

FIG. 1. Adenylate cyclase activity of cell homogenate incubatedwith lap. The total homogenate fraction of C6 glioma cells was incu-bated with lap for 5 min. Then, the reaction mixture was diluted andcentzifuged to obtain the crude membrane fraction and that fractionwas washed twice and assayed for adenylate cyclase activity withoutany additions (o) or with 10 JM isoproterenol (A), 10/M GTP (o), 10mM NaF (.), or GTP/isoproterenol (A). (A) The homogenate at 20 mgof protein/ml was incubated with Iap at various concentrations. (B)The homogenate at various concentrations was incubated with lap at2 pg/ml.

The action of NaF, however, was not affected by lap, nor wasthe basal activity estimated in the absence of these effectors.These modifications of membrane adenylate cyclase caused bydirect exposure of the broken-cell preparation to lap were thesame as those previously observed with membranes preparedfrom lap-treated intact cells (9).The more concentrated the homogenate used, the stronger

was the lap action (Fig. 1B), suggesting that factor(s) presentin the cell sap were required for lap action. When the washedmembrane preparation was used instead of the concentratedhomogenate, lap was without effect on membrane adenylatecyclase activity subsequently assayed with GTP unless ATP wasincluded in the lap treatmcnt medium (Fig. 2). Thus, ATP isone of the factors essential for lap action. Addition of the cellsap in increasing amounts enhanced lap activation of GTP-de-pendent adenylate cyclase activity progressively either with orwithout ATP, indicating that factor(s) other than ATP are alsoresponsible for lap action.NAD and ATP Requirement for the Direct Action of lap

on Membrane Adenylate Cyclase. The action of the cell sap tosupport Iap-induced activation of GTP-dependent adenylatecyclase activity was observed even with sap that had beenheated but not with sap that had been depleted of endogenousnucleotides by charcoal treatment (Table 1). NAD appeared tobe particularly important, since addition of NAD to the char-coal-treated cell sap was effective in restoring lap action. Thisaction ofNAD was not mimicked by other analogs or breakdownproducts of NAD including adenine or guanine nucleotides,nicotinamide, or ADP-ribose (data not shown). The reducedform ofNAD, NADH, orNADP could replace NAD to a limitedextent (data not shown).

Even at a high NAD concentration, addition of ATP en-hanced lap action. Probably, membrane adenylate cyclase isgradually inactivated unless the medium is fortified with ATP,as suggested by the fact that the basal cyclase activity of mem-branes is lower in the absence ofATP than in its presence (Table1 and Fig. 2). Conversely, lap action supported by NAD andATP was suppressed by a high concentration of nicotinamide(Table 1) but not by other breakdown products of NAD, suchas AMP, ADP, or ADP-ribose (data not shown).

Proc. Natl. Acad. Sci. USA 79 (1982)

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Proc. Natl. Acad. Sci. USA 79 (1982) 3131

>. _>

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Cell sap, mg/ml Iap, ug/ml

FIG. 2. NAD and ATP requirements for Iap enhancement of GTPactivation of particulate adenylate cyclase. (A) Mixtures of crudemembrane fractions (200 pg of protein) and various concentrations ofcell sap were incubated for 5 min without (o and A) or with (e andA) Iap at 10 jg/ml in the presence (A and *) or absence (o and ) of1 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 bothA andB, the particulate was separated from the reaction mix-ture, washed, and assayed for adenylate cyclase activity with 10 jiMGTP.

When the washed membranes were incubated with lap inthe presence of both NAD and ATP, the minimum concentra-tion of lap to enhance GTP activation of adenylate cyclase ac-

tivity was. 05 Ag/ml and increases in the concentration of lap

increased the enhancement progressively (Fig. 2B). lap was stillslightly effective on omission of either ATP or NAD, probablybecause of contamination of washed membranes with endoge-nous nucleotides.Time Course of lap Action on Membrane Adenylate Cy-

clase. Washed membranes were incubated with lap in the pres-

ence of NAD and ATP for periods of 2-10 min and then ana-

lyzed for adenylate cyclase activity in the presence ofGTP. Thecyclase activity is plotted as a function of time of lap treatmentin Fig. 3. GTP-stimulated adenylate cyclase activity increasedimmediately after addition of lap. The increase was initially lin-ear and the rate of this initial increase (the slope of plots at 0time in Fig. 3) was proportional to the concentration of lap from1 to 25 lLg/ml. When the anti-Iap antibody was added at 4 minof lap (5 Ag/ml) treatment, there was no further increase incyclase activity nor any decrease in activity. Thus, lap appears

to catalyze irreversible modification of membrane adenylatecyclase and progressively render the enzyme more susceptibleto GTP (and (3-adrenergic agonists). At the highest concentra-tion (25 Ag/ml) of lap, the increase in adenylate cyclase activitytended to level off within 10 min, probably reflecting satura-bility of the lap modification.

lap-catalyzed ADP-ribosylation of the Membrane ProteinofC6 Glioma Cells. The strict NAD requirement, together withthe nicotinamide-induced reversal, of the lap action suggeststhat NAD is one ofthe substrates and nicotinamide is one oftheproducts ofthe Iap-catalyzed reaction. lap treatment ofwashedmembranes was hence carried out with [a-32P]NAD. The 32pcontent of the membrane protein fractions was then analyzedby NaDodSO4polyacrylamide gel electrophoresis. As shownin Fig. 4A, a protein with a Mr of 41,000 was predominantlylabeled only when lap was present in the reaction mixture. Twominor radioactive bands (Mr, 36,000 and 29,000) were also ob-served in the presence of lap. When lap was replaced by chol-era toxin in the incubation mixture, proteins with Mr values of

200

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100 [

50

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0 2 4 6 8 10Preincubation time, min

FIG. 3. Time course of enhancement of GTP-dependent.adenylatecyclase activity by various concentrations of lap. The crude membranepreparation (2 mg of protein) was incubated with lap in 1 ml of mem-brane buffer/i mM ATP/1 mM NAD. At various times, 100 pl of me-dium was withdrawn and the particulate fraction was prepared andassayed for adenylate cyclase activity in the presence of 10 pM GTP.lap concentration added at time 0: o, none; 9, 1 qg/ml; A, 5 pg/ml;*, 25 pg/ml. Anti-lap antibody was added at 4 min to the mixture con-

taining lap at 5 ug/ml ( and A).

45,000 and 48,000/49,000 (doublet) were slightly labeled. In-cubation of membranes with both lap and cholera toxin causedlabeling of all of the protein bands labeled with either agentalone, indicating that the proteins labeled by lap are distinct

A1 2 3 4 5 6

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FIG. 4. NaDodSO4polyacrylamide gel electrophoresis of crudecell membranes incubated with radiolabeled NAD. (A) Membraneswere incubated with Iap at 25 ug/ml (lanes 2 and 5) or preactivatedcholera 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 de-scribed (21). The gel was exposed to x-ray film for 24 hr. A-: A, Mr48,000/49,000 (doublet); B, Mr 45,000; C, Mr 41,000; D M 36,000; E,Mr 29,000. Lane 6: '4C-Labeled marker proteins (Mr X 10-). (B).Mem-branes were incubated with Iap at 25 tug/ml and 125 pM (50 mCi/mmol) [carbonyl-14C]NAD (lane 1), [ribose-U-'4C]nicotinamide ade-nine dinucleotide (lane 2), and nicotinamide [U-'4C]adenine dinucleo-tide (lane 3). The gel was exposed to x-ray film for 7 days. -A, Mr 41,000band. (C) After incubation with [a-32P]NAD and Iap at 25 plg/mi, theparticulate was separated from the reaction mixture, washed, and fur-ther incubated for 2 hr at 37TC with (lane 2).or without (lane 1) snakevenom phosphodiesterase at 30 units/ml (14). The gel was exposed tox-ray film for 20 hr.

Lo-0 Ao

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3132 Biochemistry: Katada and Ui

from the substrates of the similar cholera toxin-catalyzedreaction.

lap-induced radiolabeling of the Mr 41,000 protein was alsoobserved with nicotinamide [U-14C]adenine dinucleotide andwith [ribose-U-'4C]nicotinamide adenine dinucleotide but wasnot with [carbonyl-'4CINAD (Fig. 4B). When membrane la-beled with [a-32P]NAD in the presence of lap was incubatedwith snake venom phosphodiesterase, the Mr 41,000 proteinlost most of its radioactivity (Fig. 4C). Phosphodiesterase diges-tion is known to release 5'-AMP from ADP-ribosylated proteins(14). Incorporation of radioactivity into the Mr 41,000 proteinwas suppressed by unlabeled NAD or nicotinamide at a highconcentration but not by unlabeled AMP, ADP, or ADP-ribose(Fig. 5A). These results suggest that lap catalyzes transfer oftheentire ADP-ribose moiety of NAD to the Mr 41,000 protein ofthe C6 cell membrane.

lap-induced 32p labeling was enhanced by addition of ATP(Fig. 5A), which was an essential factor for the lap enhancementof GTP-dependent adenylate cyclase activity (Fig. 2). The la-beling increased as the concentration of lap was increased from0.2 to 25 pig/ml (Fig. 5B). Addition of the anti-lap antibodyduring incubation with [a-32P]NAD prevented further labelingof the Mr 41,000 protein. These characteristics of lap-induced3P labeling are similar to those observed in Figs. 2 and 3 andTable 1 for lap-induced enhancement of GTP-dependent ade-nylate cyclase activity. Thus, ADP-ribosylation ofthe Mr 41,000protein caused by lap appears to be responsible for its abilityto enhance adenylate cyclase responses.

DISCUSSIONWe have recently reported that GTP activation of adenylatecyclase in the membrane preparation from C6 cells is enhancedby exposure of intact cells to low concentrations of lap duringculture (9). The action of lap probably results from its inter-action with the guanine nucleotide regulatory protein in themembrane adenylate cyclase system. This enhancement ofGTP activation was used, in the present study, as a measure ofdirect action of lap on the adenylate cyclase system of washed

3 4 5 6 7

iqa44 4 .-.N

B2 3 4 5

OWN 4P 40 4 - -

FIG. 5. Radiolabeling of the Mr 41,000 protein by lap. The crudemembrane fraction was labeled with (a-32P]NAD and then subjectedto NaDodSO4/polyacrylamide gel electrophoresis as described in Ma-terials and Methods. The band of the Mr 41,000 protein on an auto-radiogram is shown. (A) Labeling was carried out without lap (lane 1)orwith lapat25 ,g/ml (lanes 2-7). Additions: 1mMATP (lane 1), none(lane 2), ATP (lane 3), ATP/1 mMAMP (lane 4), ATP/1mMADP (lane5), 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-lapantibody was added 4 min after addition of lap at 5 Ig/ml. Autora-diogram was obtained after 36-hr exposure to x-ray film.

cell membranes. It is shown here that the same enhancementwas observed in cell membranes incubated with lap for a shorttime when the incubation mixture was supplemented with theconcentrated soluble fraction of the cells. The soluble factorssupporting 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 followingtwo respects. The first is the lag period so far observed on treat-ment of intact cells with lap. There was no lag in the direct ac-tion of lap on the membrane. Our detailed kinetic studies ofthe gradually developing action of lap to reverse a-adrenergicinhibition ofinsulin secretion from islet cells (6) showed that thedelayed onset of lap action represents a true lag period ratherthan a slowly reacting process. It was concluded tentatively (6)that this lag period reflected the time for gradual insertion oflap molecules into the cell membrane before reaching the siteoftheir action on the intracellular membrane surface. The pres-ent results thus support this conclusion by showing that theaction of lap started immediately on direct contact with mem-branes. It is worthy of note in this regard that the time courseof the development of lap action on membranes (Fig. 3) wassimilar to the time course for its action on intact cells that startsat 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 con-centration of lap to enhance GTP activation of adenylate cyclaseof the membranes prepared therefrom was 0.1-10 ng/ml (9).In contrast, 100-50,000 ng/ml of lap was required for directenhancement of the membrane GTP-dependent cyclase (Figs.2 and 3). It should be emphasized, however, that the concen-tration of lap required was strictly dependent on the time ofincubation in both intact cells and washed membranes. Theconcentration of lap to cause the half-maximal effect on intactC6 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 was50, 3, and 0.1 ng/ml for 6, 12, and 24 hr ofincubation (6). Thus,it is likely that, if the time of incubation of washed membranescould be prolonged to 1 to 2 hr, lap at a few nanograms permilliliter would directly enhance GTP activation of membraneadenylate cyclase toextents similar to those observed with intactcells incubated under the same conditions. Unfortunately,membrane adenylate cyclase of broken cell preparations wasso fragile, even in the presence of ATP, that prolongation ofincubation beyond 20. min caused rapid inactivation of the en-zyme and thereby obscured its susceptibility to lap. Probably,discrepancies are only apparent.

Incorporation of radioactivity from NAD into a membraneprotein was enhanced by lap when the radioactivity was locatedin the ADP-ribose moiety, but no radioactivity was incorporatedfrom the nicotinamide region. Protein that had been labeledwith [a-32P]NAD liberated 32P on hydrolysis with snake venomphosphodiesterase, indicating the presence of PP, bonding inthe labeled protein molecule. Nicotinamide, another roductofADP-ribosylation, was very effective in inhibiting [a- PINADincorporation. Thus, lap transfers the ADP-ribose moiety ofNAD to a membrane protein. It is well known that-the othertwo kinds ofprotein of bacterial origin, diphtheria (15) and chol-era (16) toxins; also catalyze ADP-ribosylation of GTP-bindingproteins.The concentration dependence, ATP requirement, and sus-

ceptibility to nicotinamide inhibition of lap-induced incorpo-ration of [a-32P]NAD into the Mr 41,000 protein were the sameas those of NAD-dependent lap enhancement ofGTP activationofmembrane adenylate cyclase. Thus, lap possesses a catalyticaction for (or induces) ADP-ribosylation ofa membrane protein,which could be responsible for its enhancement of GTP acti-

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vation (and receptor coupling) ofthe membrane cyclase in intactcells (9) or washed membranes. The time dependence of thislap action may reflect this catalytic property of the pertussisprotein. Moreover, duration of lap-induced enhancement ofGTP-dependent adenylate cyclase activity even after additionof the anti-lap antibody (Fig. 3) is consistent with irreversibleADP-ribosylation of the membrane protein.

Cholera toxin catalyzes ADP-ribosylation of one of the sub-units of the guanine nucleotide regulatory component of themembrane adenylate cyclase system (17-20). Indeed, the Mr45,000 and 48,000/49,000 proteins in C6 cell membranes werelabeled with [a-32P]NAD in the presence of preactivated chol-era toxin (Fig. 4A). This cholera toxin-catalyzed labeling wasstrictly dependent on GTP and increased on addition ofchickenerythrocyte cytosol (unpublished), in accord with previous re-ports (18, 21). Although this GTP requirement was not observedwith lap, it is possible that ATP, an essential factor for lap ac-tion, was effective in yielding GTP via phosphorylation of en-dogenous GDP. It is unlikely that the proteins ADP-ribosylatedin the presence of lap are proteolytic products of cholera toxin-specific substrates, since the labeling of its own substrates byeither toxin was not affected by simultaneous addition of theother (Fig. 4A). It is possible that the two toxins may causeADP-ribosylation of different subunits of the guanine nucleo-tide regulatory protein.

We thank Professor Osamu Hayaishi, Kyoto University Faculty ofMedicine, for his generous gift of [ribose-U-_4C]nicotinamide adeninedinucleotide and valuable discussions. We also thank Professor CharlesR. Park, Vanderbilt University School of Medicine, for his valuableadvice in preparing the manuscript. This work was supported by re-search grants from the Scientific Research Fund of Ministry of Edu-cation, Science and Culture, Japan.

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2. Yajima, M., Hosoda, K., Kanbayashi, Y., Nakamura, T., Taka-hashi, I. & Ui, M. (1978) J. Biochem. (Tokyo) 83, 305-312.

3. Ui, M., Katada, T. & Yajima, M. (1978) in International Sym-posium on Pertussis, eds. Manclark, C. R. & Hill, J. C. (NationalInstitutes of Health, Bethesda, MD), publ. no. 79-1830, pp.166-173.

4. Munoz, J. J. & Bergman, R. K. (1978) in International Sympos-ium on Pertussis, eds. Manclark, C. R. & Hill, J. C. (National In-stitutes of Health, Bethesda, MD), publ. no. 79-1830, pp.143-150.

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