Proc. Ntl. Acd. Sci. USA Vol. 75, No. 7, pp. 35-354, July 1978 Biochemistry ADP-ribosyltion of membrne proteins ctlyzed by choler toxin: Bsis of the ctivtion of denylte cyclse [GTPse/NAD/pigeon erythrocyte/poly(adp-ribose)] D. MICHAEL GILL AND ROBERTA MEREN Hrvrd University, Deprtment of Biology, 16 Divinity Avenue, Cmbridge' Msschusetts 2138 Communicted by A. M. Pppenheimer, Jr., Mrch 29,1978 ABSTRACT In the presence of ATP nd cytosolic fctor, choler toxin frgment A1 ctlyzes the trnsfer of ADP-ribose from NAD to number of soluble nd membrne-bound proteins of the pigeon erythrocyte. Evidence is presented tht suggests tht the most redily modified membrne protein (M, 42,) is the denylte cyclse-ssocited GTP-binding protein. Its modifiction by toxin is stimulted by gunine nucleotides. Adenylte cyclse ctivity increses in prllel with the ddition of ADP-ribose to this protein nd decreses in prllel with the subsequent reversl of ADP-ribosyltion by toxin nd nicotinmide. The protein is only ccessible to toxin A subunits if the erythrocytes re lysed. When denylte cyclse ctivity reches mximum, the number of ADP-ribose residues bound to this protein (bout 15 per cell) is similr to the reported number of P-drenergic receptors. The ctivtion of denylte cyclse [ATP pyrophosphte-lyse (cyclizing), EC 4.6.1.1.] by choler toxin involves the interction of some prt of the cyclse system exposed on the inner surfce of the plsm membrne with NAD, nucleoside triphosphte, nd cytoplsmic protein (1-4) nd is conveniently studied in lysed pigeon erythrocytes. The likelihood tht the NAD might function s n denosine diphosphoribose donor in toxinctlyzed rection of the type NAD+ + protein T ADP-ribosyl protein + nicotinmide + H+ ws incresed by the demonstrtions tht the ctive A1 frgment of choler toxin ctlyzes the slow hydrolysis of NAD to ADP-ribose nd nicotinmide (5) nd the trnsfer of ADP-ribose to rginine nd relted compounds (6) nd to itself (C. King, personl communiction; ref. 7). If the postulted cceptor were denylte cyclse or n ssocited membrne protein, it should be possible to demonstrte toxin-dependent trnsfer of ADP-ribose from rdioctive NAD to smll number of sites on pigeon erythrocyte ghosts. There is, however, lrge toxin-independent incorportion of ADP-ribose which ws difficult to decrese without losing the toxin response. We now report the resolution of this problem nd the demonstrtion of severl toxin-specific ADP-ribose cceptors, principlly 42, Mr membrne protein. METHODS The medium used throughout consisted of.13 M NCl,.1% sodium zide, Trsylol (protinin: FBA Phrmceuticls) t 2 kllikrein inctivtor units/ml, 1 mm N-2-hydroxyethylpiperzine-N'-2-ethnesulfonic cid (Hepes), nd NOH to give ph 7.3 t 37. Wshed nd purified pigeon erythrocytes suspended in n equl volume of this medium were lysed by rpid freezing nd then thwing. The lyste ws centrifuged t 1, X g for 5 min to seprte ghosts nd cytosol. The cy- The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U.S. C. 1734 solely to indicte this fct. 35 tosol ws mixed with well-wshed pig brin NAD glycohydrolse (Sigm) t rtio of 1 ml/1 mg, gitted t 37 for 3 min, nd then centrifuged to remove the enzyme. The ghosts were wshed three times in 2 vol of medium. There re bout 8 X 19 ghosts per ml (pcked). Portions of lyste reconstituted from 1 prt wshed ghosts nd 2 prts NAD-depleted cytosol frction were incubted for 3 min t 25 or 37 with 1 mm thymidine, 5 mm ATP,.2 vol of ctivted choler toxin (or ctivtion solution, for controls) nd [32P]NAD: either 1 MM [both phosphtes-32p]nad (1-2 mci/mmol) prepred by Robert Benjmin (8) or 1-1 MuM [phosphte in AMP-32P]NAD (5-17 Ci/mmol) (9). Toxin ws ctivted by preincubtion (1 min, 37) in 1.25% dodecyl sodium sulfte/2 mm dithiothreitol/serum lbumin (1 mg/ ml) Ḟor estimtion of incorportion nd of cyclse ctivity, the volume ws 1,l nd the incubtion ws ended by dding 1 ml of cold medium. The ghosts were recovered by centrifugtion nd resuspended. A portion of the ghost suspension equivlent to 1,ul of pcked ghosts (5 Mug of membrne protein) ws removed for cyclse ssy; the remining ghosts were wshed once more, precipitted with 5% trichlorocetic cid, filtered, nd ssyed for rdioctivity. Adenylte cyclse ws ssyed in medium including 2 mm ATP nd regenerting system but with no deliberte ddition of GTP (3). For polycrylmide gel nlysis, the ghosts of 1-3 Mul of incubtion mixture were wshed twice in 1-2 ml of medium, deep-frozen to reopen those tht hd seled during incubtion, nd wshed once more. The pellet ws gitted gently for 15 min t 37 in.2 ml of medium with dditionl.3 M NCl,.5% Lubrol PX, nd.15% cetyltetrmethylmmonium bromide [ mixture tht extrcts >95% of the rdioctive proteins].* Proteins were precipitted with 1 ml of cetone (-2, 1 hr), redissolved in 3 Ml of gel smple buffer with heting, nd subjected to electrophoresis on slb gels for 4 hr t 3 V (min gel, 7.5-15%; stcking gel, 5% crylmide.1% dodecyl sodium sulfte). Gels were stined in Coomssie blue nd destined in the presence of Bio-Rex mixed bed resin RG 51-X8, 2-5 mesh, which dsorbs ny rdioctive NAD nd ADP-ribose tht elutes from the gel. RESULTS Tble 1 lists the "bckground" nd toxin specific products formed from NAD in pigeon erythrocyte lystes. All represent the trnsfer of the entire ADP-ribose moiety of NAD, but not of nicotinmide, nd in ll cses incorportion ws decresed by unlbeled NAD but not by unlbeled ADP-ribose. * Gill, D. M. (1977) Proceedings of the 13th Joint Conference on Choler. The U.S.-Jpn Coopertive Medicl Science Progrm, pp. 195-274.
Biochemistry: Tble 1. Gill nd Meren Proc. Ntl. Acd. Sci. USA 75 (1978) 351 Acid-insoluble ADP-ribosyl derivtives formed from NAD in pigeon erythrocyte lystes Derivtives ADP-ribose/cell* Toxin-independent: Poly(ADP-ribose); nucler; very lrge nd heterogenous; inhibited by thymidine, nicotinmide, nd ATP; stimulted by ADP-ribose; stimulted nd then inhibited by DNse Up to 1, (25) Unknown soluble product; migrted rpidly on polycrylmide gels but did not form bnds; formtion ws inhibited by nicotinmide, stimulted by ADP-ribose, nd not ffected by ATP 2, (37) Mr 115, + 5 ghost protein; detergent-soluble (see Fig 1) 1 (25) Toxin-dependentt: Mjor ghost product (Mr 42,) 1,5 (25) Minor ghost products: Mr 98,, 18,, 2,, 35,, nd 2, nd others 1, (25) Cytosolic products; overll Vm,,,. = 16/cell equivlent/hr t 5 gg of toxin per ml; mjort, Mr 13,, 15,, 24,, nd 29,; minor, Mr, 22,, 58,, 67,, 71,, 92,, 15,, nd 2, nd others 1, (25) * Approximte number of residues incorported per cell equivlent during 3-min incubtion t the indicted temperture with 1,4M NAD lone or with 1,g of toxin per ml nd 5 mm ATP. t All re detergent-soluble nd form discrete bnds on polycrylmide gels (see Fig 1). ADP-ribosyltion required cytosol nd nucleoside triphosphte nd occurred to greter extent t 25 thn t 37. Those in itlics lso were found in ghost frctions. Nucler synthesis of poly(adp-ribose) initilly obscured the expected toxin-specific incorportion nd hd to be decresed to bout 1% without impiring the bility of the lyste to respond to choler toxin. Severl pproches were dopted. (i) Selective inhibitors of poly(adp-ribose) synthesis were dded: thymidine nd ATP [the ltter is lso cofctor for the toxin (3)]; 1 mm nicotinmide ws dded when studying incorportion into soluble proteins becuse it inhibits interfering rections more thn the toxin rection. (ii) The lyste ws seprted into ghost nd cytosol frctions nd the ghosts were wshed severl times. During wshing they lost much of their bility to synthesize poly(adp-ribose). (Mii) The NAD in the cytosol ws hydrolyzed by incubtion with n insoluble NAD glycohydrolse tht ws then removed by centrifugtion. This technique llowed us to replce endogenous NAD with low mount of rdioctive NAD without diluting the cytosol or decresing its bility to support the ctivtion of denylte cyclse. By contrst, removl of endogenous NAD by dilysis, dsorption on chrcol, or column chromtogrphy hs generlly resulted in mjor inctivtion. Endogenous ATP is lso hydrolyzed during the preincubtion. Thus, lyste reconstituted from wshed ghosts nd NAD glycohydrolse-treted cytosol is lmost totlly refrctory to choler toxin but is restored to its originl sensitivity upon the reddition of both NAD nd ATP. With ech nucleotide t 5 mm nd n incubtion of 3 min t 37, denylte cyclse ctivtion is detectble with 1 ng of choler toxin per ml nd complete with 1 ng/ml. Under such conditions, one toxin molecule cn ctlyze the incorportion of more thn 5 ADP-ribose residues per hour. For the present experiments we hve used NAD t 1,M or less nd correspondingly more toxin (1-1,ug/ml); the sme toxin-specific rdioctive products were formed s with high NAD nd low toxin. The toxin-specific incorportion is 2-3 times the totl bckground incorportion of ADP-ribose. (iv) Much of the residul poly(adp-ribose) is lost upon extrcting the ghosts with detergent nd frctionting the extrct on polycrylmide gels. The smll mount of poly(adp-ribose) removed by detergent fils to enter the min gel nd the only toxin-independent rdioctive bnd found represents protein of bout 115, Mr. The toxin-dependent products re esily recognized (Fig. 1). Anlysis of the Toxin-Specific ADP-Ribosyl Products. Unexpectedly, choler toxin ws found to ctlyze the trnsfer of ADP-ribose to mny intrcellulr cceptor molecules both in the membrnes nd in the soluble frction (Tble 1; Fig 1). The ptterns observed were reproducible nd did not vry substntilly with time or with the ddition of protese inhibitors. The sme bnds were seen when the totl (deoxyribonuclese-treted) ghost pellet ws frctionted, rther thn detergent extrct, but the resolution ws inferior. The rdioctive products formed only nd lwys under conditions tht resulted in ctivtion of denylte cyclse (Fig. 1). Thus, ADP-ribosyltion of ll or ny required the presence of thiol-reduced subunit A or of choler toxin from which A hs relesed by preincubtion with dodecyl sodium sulfte (4) nd ws prevented by ntitoxin or nti-subunit A but not by ntisubunit B. ADP-ribosyl trnsfer ws stimulted by nucleoside triphosphte (e.g., ATP, UTP, 5'-denylylimidodiphosphte), ws inhibited by certin methylxnthine nlogs of ATP, nd ws prevented if endogenous ATP ws hydrolyzed by prior tretment with n ATPse. Lbeling of the ghost-bound products depended on the presence of cytosol in high concentrtion. The mount of product ws roughly proportionl to the -115, - 98, -m- -42, - 24, - 22 2 3 4 5 6 7 8 9 1 FIG. 1. Gel nlysis (utordiogrm) of rdioctive products. Incubtion: 5 mm ATP, 1 mm thymidine, 1 mm nicotinmide, 5 MM NAD, 1,Mg of ctivted toxin per ml, in 13,Ml; 3 min, 37. Lnes: 1-7, ghosts; 1, no nicotinmide; 2, no dded ATP (endogenous ATP only); 3, no cytosol; 4, toxin not preincubted with sodium dodecyl sulfte nd dithiothreitol; 5, no thymidine; 6, control; 7, toxin neutrlized by nti-subunit A. Lnes 8-1: 3 Ml of the soluble frction; 8, control; 9, incubtion t 25; 1, incubtion without ghosts.
352 Biochemistry: Gill nd Meren 1 2 3 Adenylte cyclse ctivity, pmol cyclic AMP/,l/hr FIG. 2. ADP-ribose incorportion nd denylte cyclse ctivities t different toxin concentrtions. Conditions s in Fig. 1 except 1 4M NAD nd vrible toxin concentrtions, s mrked (,ug/ml). Rdioctivity in the totl ghost pellet ws corrected for the incorportion without toxin. The intersection of the two portions of the curve (rrow) here represents bout 2 ADP-ribose residues per ghost. denylte cyclse ctivity, provided tht the ctivtion ws not complete. Extrpoltion generlly reveled tht n verge of 15-3 ADP-ribosyl residues would be ssocited with ech ghost if every copy of the cyclse ws ctivted (Fig. 2). Continued incubtion fter this point (or higher toxin or NAD concentrtion) results in further ADP-ribose incorportion without further increse (usully with slight decrese) in cyclse ctivity. It ws obviously importnt to determine which, if ny, rdioctive product ws responsible for the ctivtion of denylte cyclse. We estblished first tht none of the cytosolic cceptors ws relevnt lthough some of them (Tble 1) tended to ssocite with the ghost frction nd my represent significnt proportion of the totl ghost-ssocited product, especilly t high toxin nd NAD concentrtions or with extended incubtions. The ssocition seems to be incidentl becuse the mount in the ghost frction vried considerbly with experimentl conditions nd, upon post-incubtion in fresh medium, some of the mteril ws lost from the ghost frction without corresponding decrese in cyclse ctivity. In ny cse, denylte cyclse ws not ffected when untreted ghosts were incubted with toxin-treted cytosol in the presence of ntitoxin or of NAD glycohydrolse, even though some trnsfer of ADP-ribosylted proteins occurred. ADP-ribosyl A1 ws not significnt product under our usul conditions: no rdioctive mteril ws dsorbed by nti-subunit A linked to Sephrose beds. However, it ws detectble fter incubtion t very high toxin concentrtions nd, s with ntive A1, tended to dsorb to ghosts (see Fig. 3, lne 2). Importnce of 42, Mr Peptide. The mount of the dominnt ghost-bound product, 42, Mr, ws determined fter utordiogrphy by ssying the rdioctive bnd excised from dried gels. The remining portions of the gels were lso ssyed to determine the overll recovery of rdioctive mteril. We hve not been ble to demonstrte complete recovery consistently, which hs mde ccurte quntittion difficult, but we could drw the following conclusions from those gels in which we could ccount for 9% or more of the rdioctivity of the originl smple. (i) Under limiting conditions, the ADP-ribosyl 42, Mr protein represents 5-6% of the totl toxin-specific incorportion into ghosts nd prllels the ctivtion of denylte cyclse. (ii) The mount present t mximum cyclse ctivity, determined by n extrpoltion similr to tht in Fig. 2, is bout 15 per cell, less thn.1% of the Proc. Ntl. Acd. Sci. USA 75 (1978) membrne protein; this mount is similr to the estimted number of f3-drenergic receptors nd thus possibly to the number of denylte cyclse molecules in pigeon erythrocyte (1). (iii) Fewer thn 2 copies of ny other ghost protein re lbeled under limiting conditions. (iv) Further incubtion results in the continued ddition of ADP-ribose residues to the 42, Mr bnd (see Discussion). (v) The 42, Mr protein remins the dominnt cceptor to high toxin concentrtions but is eventully sturted. It is evidently prticulrly efficient cceptor. Thus, for exmple, the 98, Mr membrne protein is evidently much more bundnt but is only lbeled to greter extent t extrordinrily high toxin concentrtions (compre Fig. 1 with lne 4 in Fig. 3). The 42, Mr protein ws entirely soluble in the nonionic detergents Lubrol PX nd Triton X-1, suggesting tht it is membrne-bound. This ws confirmed by frctiontion. After ghosts hd been disrupted with Yed press, the rdioctive protein ws ssocited with 1, X g (membrne) pellet, in contrst to poly(adp-ribose) which ws found only in low-speed (nucler) pellet. The protein must be locted on the inner fce of the cell membrne becuse it cnnot be lbeled from outside the erythrocyte. To estblish this, intct erythrocytes were suspended in cytosol obtined from other cells nd incubted with ATP, [32P]NAD, nd toxin A subunits. In order to void cell lysis, the A subunits were pretreted only with 5 mm dithiothreitol but not with dodecyl sodium sulfte. As shown in Fig. 3, there ws no lbeling of the 42, Mr protein nd little lbeling of ny protein, unless the cells were lysed, despite the reltively enormous concentrtion of subunit A used. [Although there is report to the contrry (11), we do not find tht isolted subunit A significntly increses the denylte cyclse of intct erythrocytes. ] On incubtion of lbeled ghosts with trypsin t 5,tg/ml for 1 min, the 42, Mr protein ws completely converted to 4, Mr peptide with minor mounts of rdioctive peptides of 28,, 19,, nd 17, Mr. The filure to digest the ht 98o 42, 24, _pg 22, A, 2 3 4 FIG. 3. Intct erythrocytes were suspended in cytosol nd incubted with 8 MM [32P]NAD, 5 mm ATP, nd isolted toxin subunit A preincubted with 5 mm dithiothreitol, to finl concentrtion of 1 Mg/ml, equivlent to bout 3 Mug of toxin per ml. After 3 min t 37 the externl cytosol ws removed by wshing, nd the erythrocytes were lysed nd seprted into internl cytosol (lne 1, no bnds visible) nd ghost (lne 2) frctions. Lnes 3 nd 4 represent cytosol nd ghost frctions from prllel incubtion of the sme volume of lysed erythrocytes incubted with ATP nd subunit A. In this cse the rdioctive NAD ws diluted bout 1:14 by the erythrocyte NAD. The detergent-soluble product represented bout 12 ADP-ribose residues per ghost (intct) nd 2, per ghost (lysed). The bnd in lne 2 mrked Al ppers to be some ADP-ribosyl-Al.
protein more thoroughly my result from its ssocition with the membrne. The 42, Mr protein migrted slightly more rpidly on polycrylmide gels when it hs not been pretreted with thiol reducing gent. Such behvior is usully tken to indicte tht one or more intrchin disulfide bonds re present nd re responsible for some conformtionl constrint. There re evidently no interchin disulfides. Reversl of Incorportion of Cyclse Activtion. The ADP-ribosyltion of ghost proteins could be reversed by incubting the wshed modified ghosts with ctivted choler toxin (or A) nd nicotinmide. Both re essentil. Reversl ws ided by wshing the ghosts first nd by dding NAD glycohydrolse to decrese the NAD level. Even then, high concentrtion of nicotinmide (1 mm > 1 mm) ws required. Presumbly the equilibrium is considerbly bised towrd the forwrd rection s is the cse with other ADP-ribosyl trnsfers. Reversl lso ws ided by decresing the ph. This suggests tht proton -I ) z ) * Biochemistry: 1K 8K 6K 4K 2k- Control A? - - Gill nd Meren 1 mm fluoride @ 2 4 6 Incorported ADP-ribose, pmol/1 Ml ghosts FIG. 4. Decrese of bsl denylte cyclse ctivity nd increse in fluoride-stimulted ctivity upon reversl of ADP-ribosyltion. A reconstituted lyste ws prtilly intoxicted in the presence of 1 MM [32P]NAD (1 sg of toxin per ml, 25, 3 min, no nicotinmide). Portions were then diluted in 2 ml of buffer nd centrifuged. The pellets, contining 85 Ml of ghosts, were resuspended in 1 Ml of cytosol to (finl concentrtions) 5 mm ATP, 1 mm nicotinmide,.5 unit of NAD glycohydrolse per ml, morpholinopropnesulfonic cid to ph 6.4, nd 1,g of toxin per ml. Incubtion under these reversl conditions ws for 3 min t 25. The ghosts were recovered nd wshed well. Smll portions were ssyed for denylte cyclse ctivity in the bsence nd presence (upper line) of 1 mm sodium fluoride (ordinte). Acetone precipittes of detergent extrcts of the remining ghosts were ssyed for rdioctivity (bsciss). &, Control ghosts incubted with NAD but without toxin;, toxintreted ghosts before reversl. ADP-ribosyltion reversed by incubtion in complete medium (), without nicotinmide (N), with no dditionl toxin (o), with no dditionl ATP (), or t (-). O Proc. Ntl. Acd. Sci. USA 75 (1978) 353 I-BAND 3 -TUBULIN N 1-742,~42 _v-; AC T I."...2_3, > Hf STONES _- GLOBIN 1 2 3 4 5 6 7 8 FIG. 5. Specific removl of [32P]ADP-ribose residues from the 42, Mr protein. Conditions of lbeling nd reversl were s in Fig. 4. Extrcts of 8 Ml of ghosts were frctionted; the ppernce fter stining is shown on the right Autordiogrm lnes: 1, full; 2, no NAD glycohydrolse or morpholinopropnesulfonic cid; 3, no morpholinopropnesulfonic cid; 4, only 1 mm nicotinmide; 5, no NAD glycohydrolse; 6, no ATP; 7, no toxin; 8,. is involved; thus: nicotinmide + H+ + ADP-ribosyl proteins toxin A1 -* NAD+ + proteins. As the ADP-ribose is removed, the denylte cyclse ctivity decreses from its elevted level. Tht this decrese is not due to nonspecific inctivtion ws shown by the ccompnying increse in fluoride-stimulted cyclse ctivity, property tht ws decresed by choler toxin (Fig. 4). Polycrylmide gel nlysis of the ghosts confirmed tht much ADP-ribose ws removed from the 42, Mr protein under ny condition tht resulted in ADP-ribose removl in generl (Fig. 5). Involvement of GTP. Enomoto nd Gill (12; cf. 13) recently found tht denylte cyclse ctivtion by choler toxin in vitro is somewht dependent upon GTP (distinct from ny effect of GTP during the subsequent cyclse ssy) nd tht the dependence is most pprent when whole cytosol is replced by the mcromoleculr frction obtined on Sephdex G-25 column. Fig 6b shows such dt, nd Fig 6 shows tht 1 mm GTP nd, even better, 5'-gunylylimidodiphosphte lso stimulte ADP-ribosyltion even in the presence of n excess of ATP. The rtes of toxin-ctlyzed ADP-ribosyltion nd of denylte cyclse ctivtion were not ffected by 1 mm sodium fluoride or.1 mm epinephrine. DISCUSSION We hve shown tht choler toxin ctlyzes the trnsfer of ADP-ribose from NAD to number of membrne-bound proteins under circumstnces tht result in the ctivtion of denylte cyclse. It is resonble to suppose tht ADP-ribosyltion of one of these proteins ccounts for the cyclse ctivtion nd tht the event tht hs been detected here in vitro occurs lso in intct cells fter the pssge of frgment A1 from receptor-bound holotoxin to the cell interior. The following observtions mke it ttrctive to suppose tht the 42, Mr protein is the physiologiclly relevnt trget in terms of denylte cyclse ctivtion nd, furthermore, tht it my be the GTP binding component of the denylte cyclse system. (i) correltion between the increse in denylte cyclse ctivity nd the extent of ADP-ribosyltion of this protein; (ii) correltion between the decrese in denylte cyclse ctivity nd the extent of removl of ADP-ribose residues on incubtion of the modified membrnes with toxin nd nicotinmide; (iii) n pproximte correspondence between the number of copies of this protein per cell tht need to be ADP-
354 Biochemistry: Gill nd Meren D. Oc -o '._ OL *-t E -L. _ -Q f._ >u 2 1 4 _ 3_ 2 i n1 X b l;k? I Gpp(NH)p AA A GPp(NH)p GTP - L _.3.6 1 1.6 2.5 4 Toxin, gg/ml FIG. 6. Stimultion of choler toxin's ction in vitro by gunie nucleotides. Incubtion: lyste reconstituted from wshed ghosts nd the mcromoleculr frction of cytosol prepred on Sephdex G- 25-15,5 mm ATP, 1 mm thymidine, 1.2MuM NAD, toxin s shown, with no ddition (), 1 mm GTP () or 1 mm 5'gunylylimidodiphosphte (Gpp(NH)p) (C) t 25 for 3 min. Adenylte cyclse ctivtion ws incomplete. Seprte portions of wshed ghosts were extrcted with detergent, precipitted with cetone nd ssyed for rdioctivity () or wshed twice to remove gunine nucleotides nd ssyed for denylte cyclse ctivity (b). Bsl cyclse ctivity (2 pmol/;tl per hr) hs been subtrcted. The upwrd displcement of the Gpp(NH)p curve represents cyclse ctivtion by the nucleotide itself. ribosylted to chieve mximl cyclse ctivity nd the reported number of fl-drenergic receptors per cell (1) nd the lck of such correspondence for ny other protein; (iv) size identicl to tht reported for the cyclse-ssocited GTP binding protein (1); (v) loction on the inner surfce of the plsm membrne; (vi) prior evidence tht the GTP binding subunit [or possibly two subunits (14)] modultes denylte cyclse ctivity nd is required for cyclse-ssocited GTPse function (15) [it hs been proposed tht the ctive form of denylte cyclse, with GTP bound, reverts to inctivity when the GTP is hydrolyzed nd the split products re relesed; choler toxin, by inhibiting the GTP hydrolysis is thought to trp denylte cyclse in its ctive stte (15-18)]; (vii) stimultion by gunine nucleotides of ADP-ribosyltion nd of denylte cyclse ctivtion (this could men tht the trget protein is more efficient substrte for the toxin when GTP is bound to it; indeed, becuse trce of ghost-bound GTP my hve been present in ll our experiments, it is possible tht the trget protein my be toxin substrte only when bound to GTP). Persistence of the bound GTP might explin the filure of the modified protein to bind to GTP ffinity mtrices becuse we were unble to detect specific binding of rdioctive product to Sephrose 6B-NH-(CH2)6 CO-CNH NHpppG, lthough similr mtrix dsorbs the unmodified cyclse-ssocited GTP-binding protein (1). Proc. Ntl. Acd. Sci. USA 75 (1978) After bout 15 ADP-ribose residues per cell hve been incorported into the 42, Mr protein, it my continue to ccept ADP-ribose without further increse in denylte cyclse ctivity. Thus, if it is indeed responsible for the cyclse ctivtion, it is simplest to regrd it s dissocible component of the cyclse system, present in excess over the ctlytic nd ctecholmine receptor components. The physiologicl effects of the other ADP ribosyltions observed re not cler. The possibility is not excluded tht modifiction of one or more secondry trgets my hve cellulr consequences, nd even dditionl effects on denylte cyclse. However, becuse these modifictions re reltively slow, they my be inconsequentil unless they shre the peculir elegnce of the toxin's effect on denylte cyclse, in which the inhibition of one property (GTPse) disproportiontely increses nother ctivity (cyclic AMP synthesis). As is the cse for the mjor trget, GTP stimultes the ADP-ribosyltion of severl (but pprently not ll) of these secondry trgets s if choler toxin recognizes some feture common to the nucleotide binding sites of vrious GTP-binding proteins. This possibility is prticulrly interesting in view of the remrkble similrities between the ctions of choler nd diphtheri toxins. Frgment A1 of diphtheri toxin ctlyzes the trnsfer of ADP-ribose from NAD to EF2, the eukryotic polypeptidyl-trna trnslocting fctor, which is protein tht binds GTP. After EF2-GTP binds to ribosomes, the GTP is split. This work ws supported by Ntionl Institutes of Helth Grnt Al 1383. 1. Gill, D. M. & King, C. A. (1975) J. Biol. Chem. 25, 6424-6432. 2. Gill, D. M. (1975) Proc. Nt!. Acd. Sci. USA 72,264-268. 3. Gill, D. M. (1976) J. Infect. Dis. 133, S55-S63. 4. Gill, D. M. (1976) Biochemistry 15, 1242-1248. 5. Moss, J., Mngniello, V. C. & Vughn, M. (1976) Proc. Ntl. Acd. Sci. USA 73,4424-4427. 6. Moss, J. & Vughn, M. (1977) J. Biol. Chem. 252, 2455-2457. 7. Trepel, J. B., Chung, D.-M. & Neff, N. H. (1977) Proc. Ntl. Acd. Sci. USA 74, 544-5442. 8. Colowick, S. P. & Kpln, N.. (1957) in Methods in Enzymology, eds. Colowick, S. P. & Kpln, N.. (Acdemic, New York), Vol. 4, p. 852. 9. Ued, K. & Ymmur, H. (1971) in Methods in Enzymology, eds. McCormick, D. S. & Wright, L. D. (Acdemic, New York), Vol. 18B, pp. 6-66. 1. Pfeuffer, T. (1977) J. Biol. Chem. 252,7224-7234. 11. vn Heyningen, S. & King, C. A. (1975) Biochem. J. 146,269-271. 12. Enomoto, K. & Gill, D. M. (1978) J. Suprmol. Struct. in press. 13. Moss, J. & Vughn, M. (1977) Proc. Nt. Acd. Sci. USA 74, 4396-44. 14. Ld, P. M., Welton, A. F. & Rodbell, M. (1977) J. Biol. Chem. 252,5942-5946. 15. Cssel, D. & Selinger, Z. (1977) Proc. Nti. Acd. Sci. USA 74, 337-311. 16. Flores, J. & Shrp, G. W. G. (1975) J. CGn. Invest. 56, 1345-1349. 17. Levinson, S. L. & Blume, A. J. (1977) J. Biol. Chem. 252, 3766-3774. 18. Johnson, G. L. & Bourne, H. R. (1977) Blochem. Blophys. Res. Commun. 78, 792-798.