The Glucagon-sensitive Adenyl Cyclase System in Plasma Membranes of Rat Liver 1~. EFli`ECTS OF GTJANYI, SUCLEOTIDES ON BINDING OF 9-GLUCAGON (Received for publication, October 7, 1970) MARTIN RODBELL, H. MICHIEL J. KF~ANS,* STEPHEN L. POHL,AND LUTZBIRNBAUYER From the Se&m on iMembrane Regulation, Laboratory of Nutrition and l3'ndocrinoJogy, .Valimal Institute of Arthritis and Metabolic Diseases, Bethesda, Maryland 20014 SUMMARY Studies have been made of the effects of nucleotides on binding of 1*61-glucagmm at its specitlc binding sites in plasma membranes of rat Ever. GTP and GDP, equally and at a minimal concentration of 0.05 m, stimulate the rate and degree of dissociation of bound labeled hormone, decrease uptake of glucagon by the membranes, and decrease the aflinity of the binding sites for glucagon. These effects of the nucleotides are concentra- tion-dependent, reversible, and rapid in onset. Divalent metal ions are not required for the actions of the nucleotides which act equally at 0' or 30'. 5'.Methylene guanylyl-di- phosphonate, a nonphosphorylating analogue of GTP, mimicked the effects of GTP or GDP on glucagon binding although at 100 times the concentration of the natural nucleo- tides. Based on these observations and the finding that the nucleotides do not act competitively with glucagon, it is sug- gested that GTP or GDP regulate glucagon binding by an allosteric type of action. This action of the guanyl nucleo- tides is inhibited by a sulfhydryl reagent (P-chloromercuri- benzoate), which also inhibits binding of glucagon. Sodium fluoride, which stimulates adenyl cyclase activity in liver membranes, has no effect on either the binding of glucagon or on the actions of guanyl nucleotides on this process. ATP, ADP, UTP, and CTP act similarly to the guanyl nucleotides on the glucagon binding process but only at con- centrations greater than 0.1 mx Cyclic 3',5'-GMP, 5'. GMP, and the corresponding adenine nucleotides are inac- tive on the glucagon-binding process. In the previous study (I) correlations were found between binding of L2hI-glucagon to specific biding sites in rat liver plasma membranes and activations of adenyl cyclase by the hormone, suggesting that the specific binding sites are com- ponents of the adenyl cyclase system. It was also found that EDTA stimulated dissociation of hound labeled glucagon. This * ltecipient of a fellowship from Setherlands Orgnnization for PIIre Scientific Research (Z.W.O.), 1969-1970. phenomenon plus the marked temperature dependence of the binding process suggested that the reaction between hormone and binding site is complex and may be influenced by other factors. During the cctilrse of investigating whether binding of glucagon may be affected by ATP, the substrate for adenyl cyclase, and by other components of the medium u& for assaying adenyl cyclase activity, it was d&covered that 0anyl nucleotides, at extraordinarily low concentrations, altered the binding of 1*61- glucagon. These findings are detailed in the present report. EXPERIMENTAL PROCED- Only those materiaIs and methods not described in the pre- ceding papers (l-3) are documented in the present communica- tion. Materials-ATP, ADP, AMP, and GRIP were obtained from Sigma; GTP, GDP, UTP, and CTP from P-L Laboratories; cyclic 3',5'-AMP from Calbiochem; q&c 3',5'-GMP from Schwarz BioResearch. 5'-Guanylyl methylene diphosphonate was purchased from Miles Laboratory; pchloromercuribenzoate from Sigma. `GTP-a-**P was obtained from International and Nuclear Corporation. Measurement of Hydrolysis of GTP by .Vuclwfi&ws in Plasma Mem&neePlasma membranes (25 fig of protein) were incu- bated at 30" in medium containing 2.5F; bovil:e serum albumin (Fraction V), 20 my Tris-HCl, pH 7.6. and 50 PM GTP+P (specific activity, 60,000 cpm per nmolei in a final volume of 0.125 ml. After 2 min of incubation, the reaction was stopped by the addition of 1 ml of 0.16 M prchloric acid containing 2 fimoles Of Pi. The mixture was al1oKed to stand for 10 min at 0" and was then centrifuged at 2000 rpm in .sn International refrigerated centrifuge. "Pi liberated during incubation was isolated by the following modification of the method of Sugino and Miyoshi (4). To 0.5 ml of the eupm.stant fluid was added 0.75 ml of a solution containing 8 no triethylsmine-HCl (pre- pared by titrating a 0.2 M solution with 1 .v IICl to pH 5.0), 6.4 mnr ammonium molybdate, and 16 111~ prchlaric acid. After standing in the cold for 10 min the precipitate of triethylamine- phosphotnolyhdate (4) was collected on 0 45 p llillipore filters and washed with 1 ml of ice-cold wafer. The 61~s were allowed to dry in counting vials and were dissolved slonp n-ith the precipi- tate in 1 ml of acetone. **Pi was determmed in 15 ml of Bray's 1872 T.\VLE I ~,~~~ccls of AI'Y, Mg++, and A 7'1'-rt,yc,rdprnlin!l u/stern (crealine kinuse-phosphorrenfinc) otl ccpldir oj ylueayon 611 plasma membranes lncilbations were carried out, tit 30" for 20 min. The standard mpdiunl for incubation contained, in 0.125 ml, 25 pg of membrane protein, I mu EDTA, 2.5!Y, albumin, and 20 mM Tris-HCI, pi1 7.6. The complete medium contained, in addition, 5 mM MgCl~, 3.5 mM ATI', 20 rn~ phosphocreatine, and I mg of creatine phosphokirlase (Sigma, 44 units per mg) per ml. Both media contained 4.2 IBM IzsI-glllcagon (lOs cpm per-pm&?). Separation of bound from free labeled glucagon was carried alit a~+ described elsewhere (1). Incubation medium Standard . 1.05 Complete. _ . . 0.50 -ATP-regenerating system.. . . 0.56 -MgClr . . . 0.52 -ATP. . . . 1.33 scintillation fluid (5) in a liquid scintillation counter. Based on the specific activity of labeled GTP, the quantity of radioactivity was converted to picomoles of Pi liberated. Activities of the nucleotidase (or nucleotidases) are expressed as nanomoles of Pi liberated per min per mg of membrane protein. Protein was determined by the method of Lowry et al. (6), as modified in the previous study (1)) using crystalline bovine albumin as standard. RESULTS Efects of ATP on Binding of Labeled Glwagm to Liver Plasma Ilfembranes-Addition of 3.2 rnM ATP, 5 mab MgC&, and an ATP-regenerating system (creatine kinase and phosphocreatine) to the standard incubation medium (2.5% albumin, 1 mM EDTA, 20 mM Tris-HCl, pH 7.6) used in the previous binding studies (1) forms the complete medium used for assaying adenyl cyclase in liver plasma membranes (2). Binding of 1*61-glucagon to the membranes was reduced by 50% in the complete medium com- pared to the standard medium, as shown in Table I. This re- duction was caused by ATP since its omission from the complete medium resulted in restoration of binding to levels observed in the standard medium. E$ects of Nucleotides on liptake or Dissociatima of Bound Lu- beled Hormone from Liver Jfembranes-Under adenyl cyciase incubation conditions, TTP, CTP, and GTP, at 3 mM, decreased uptake of glucagon by liver membranes and stimulated dissocia- tion of bound labeled glucagon (Table II). It is seen that GTP was the most effective nucleotide, causing complete dissociation of bound hormone within 15 min of incubation. In subsequent experiments it was found that ATP and the pyrimidine nucleo- tides did not affect either uptake or dissociation of glucagon from the membranes at concentrations less than 0.1 mx. The effects of GTP and Ai'l`l', at varying concentrations, on the dissociation process are described iu Fig. I. GTP stimulated dleociatinn of ~~UC:LgOn at, x concpntrntion as low as 0.05 j.&bl whereas the mini- mal effective concentration of ATI' ww betweeu 100 PM and 500 MU. It will also be noted that 0.05 11lN GTP stimulated complete release of bound labeled glucagon. NSO t.&cd WPI`C the cffccts of v:\rious known mctabolitc~ of (.;TI' on the dissoriat~ion process (Table III). At the .sanre COW centration, 1 .O MU, Gl)P was as effective as GW in stimul:lt.ing T\IILF: II and dissociulion of bo?cntl In6clcd qlucngon from liver memhmncs Plasma mnrnbmncs (75 pg of proteiil) were incubated in 0.375 ml of complet~e medium (described in legend to Table I) containing *z'I-glucagon (4.5 I\M) with the indicated nucleot,ides either omitted or added at final concentration of 3.0 mM. In studies of uptake of glueagon, incubations were at 30" for 15 min. In studies of dissociation of bound labeled glucagon, incubations were car- ried out as above but with no added nucleotides during the first 15 min of incubation. At 15 min, duplicate 50-~1 aliquots were withdrawn for assay of bound glucagon, and 125~1 aliquots were added to 10~1 of a solution containing unlabeled glucagon and the indicated nueleotides to give final concentrations of 5.0 pu and 3.0 mM, respectively. Incubations were continued for 15 min at 30", after which 50-d aliquots were again taken for assay of bound labeled glucagon. Bound labeled glucagon was assayed by Method A described in the previous report (1). Calculations of percentage of labeled glucagon dissociated were made as described in legend to Fig. 1. Results are the average of two experiments i S.E.M. Additions uptake of gbcagon Bound `Wglucagon dissociated #?molu/mg p7otcin % None 1.16 i 0.03 49 f 2 CTP 0.75 * 0.04 65 zt 3 UTP 0.71 i 0.04 75 zt 4 ATP 0.59 f 0.02 75 f 4 GTP 0.43 i 0.02 100 f I I I I t I I I I I -9 -6 -7 -6 -5 -4 -3 -2 -i GTP or ATP ( IogfM]) Fro. 1. Effects of varying concentrations of GTP and ATP on dissociation of 9-glucagon from plasma membranes. Incuba- tions were carried out in two stages. In the first stage, plasma membranes (150 rg of protein) were incubated at 30" in a final volume of 0.75Oml of incubation medium containing 2.5y0 albumin, 1 mM EDTA, 4 nM `**I-glucagon (10' cpm per pmole), and 20 rn36 Tris-HCI, pH 7.6. After 15 min, duplicate 50-J aliquots were taken for measuring bound labeled glucagon as described pre- viously (1). In the second stage of incubation, GTP or ATP were added simultaneously with unlabeled glucagon (final con- centration, ~.O'JJM) to give the indicated concentrabions. After 15-min incubation at 30", 50-~1 aliquots were taken for measuring bound labeled glucagon as above. The percentage of bound labeled glucagon dissociated dnring the second stage was calcu- lated from the difference between the amount of bound labeled glucagon in the first and second stages of incubation as desrribed previously (1). the dissociation of bound labeled glucagon; other experiments &owrd thcsc nuclcotidcn to Ix equally etIecti\r :lt. 0.05 ~31, (`yrlic: 3',5'-GRIP and 5'-G111' did not alter dissociation of bound ~lucagon or uptake of the labrled hormone by liver mem- 1874 G'lucapn-sensitive Ade?ql C~&s~. IV Voi; 246, No! ti; banes. III similar cslwrhcnts it W:IS found that A'I'P and mr,, at 3 mM, exertr>tl ecluivalrnt c+Iects on dissociation of hound labeled glucago~~ ; cyclic 3' ,5'-MI 1' and 5'-AMP, at 3 mM, did not alter binding of labeled glucngon. Efleck of GTP on Time Course oj Diswciation of Bound Gluca- gon-The stimulatory effects of GTP, at concentrations ranging from 0.1 PM to 0.1 mu, on dissociation of bound labeled glucagon were rapid in onset; significant stimulation was observed within 1 min of the addition of GTP to the incubation medium (Fig. 2). Although initial rates were too rapid to measure accurately, it can be seen that both the rates and degree of dissociation were dependent upon the initial concentration of GTP. The effects of GTP OJI dissociation of bound labeled glucagon were not dependent upon the presence of unlabeled glucagon in the incubation medium. As shown in Fig. 3, omission of unla- beled glucagon (5.0 PM) reduced o~dy the extent of dissociation TABLE III Efecla of GTP, GDP, GMP, and q&c J',Q'-GMP on dissociation of bound 1z51-glucagon from liver membranes Plasma membranes (75 rg of protein) were incubated in 0.375 ml of medium contaiuing 2.5% albumin, 1 mM EDTA, 4.5 nM W- glucagon (10" cpm per pmole), and 26 mM Tris-HCl, pH 7.6. In- cubations were for 15 min at 36". Duplicate 56-d aliquots were withdrawn for assay of bound glucagon (Method A in previous study (l)), followed by addition of 10 ~1 of a solution containing unlabeled glucagon and the indicated nucleotides to give final concentrations of 5 PM and 1 &M, respectively. Incubations were continued for 15 min at 30", after which 50-~1 aliquots were again assayed for bound labeled glucagon. Additions None 5'-GMP Cyclic 3',5'-GMP GDP GTP Bound u*I-+cagon diswxiatcd % 37 35 36 6.5 63 in 3 min of inrubat.ion; the difference from that observed in the, presence of unlabeled ghCagoll lnobably reflects reassociation of.' labeled glucagon at its binding sites. Eflects of lVatious Concentrations of GTP on Levels of Bound' Glucagon-;-As was noted previously for the effects of ATP (Table, I), GTP also decreased the levels of glucagon taken up by liver. membranes. In Fig. 4, it is seen that the effects of 10~ concen- trations of GTP on uptake of labeled glucagon were less marked- than on dissociation of bound hormone. However, the eoncen-- tration of GTP required for minimal effects on uptake and disso- ciation were essentially the same, about 0.05 PM. Eflects of GTP on Afinity of Bindins Sites jar Clucagon-GTP' (1.0 FM) did not modify the number of binding sites for labeled glucagon but shifted the concentration of glucagon required for half-maximal binding from 2.5 nM to about 6.0 no (Fig. 5). Similar studies were not carried out at higher concentrations of CTP, although it is evident from the marked effects of 1.0 mad GTP on the uptake of glucagon (Fig. 4) that the apparent affinity of the binding sites for glucagon is decreased further at this concentration of the nucleotide. Effects of GTP on Time Course of Binding in Presence and Absence of EDTA-Studies of the effects of GTP on binding were carried out in the presence of EDTA (1 .O mM) in a11 of the studies reported thus far. As shown in Fig. 6,l NM GTP, in the presence or absence of EDTA, decreased the initial uptake of glucagon. However, in the absence of EDTA, the inhibitory effects of GTP on uptake of glucagon were not sustained. After 10 min of incu- bation, uptake of glucagon returned to levels approaching that attained in the absence of GTP; in the presence of EDTA the effects of 1 PM GTP were sustained for at least 25 min. It will be noted that GTP decreased the time at which a constant level of bound glucagon is attained (5 min) compared to the control (about 15 min). EDTA did not alter the time course of uptake of glucagon or the amount bound. E@ts of EDTA on Hydrolytis of GTP by Nuckotidase in Plama MemImznes-Plasma membranes of rat liver have been shown to centain magnesium or calcium ion-dependent nucleo- 3 MINUTES to-6 10-S Kr4 10-3 GTP (Ml Fm. 2. (Iefl). Effects of GTP, at various concentrations, on the unlabeled glucagon (5 PM, final concentration) and the indicated time course of dissociation of l*sI-gliicagon from plasma mem- concentrations of GTP. Incubation time was 3 min. branes. Experiments were carried out in the same manner and FICJ. 4 (riphl). Effects of various concentrations of GTP on under the incubation conditions described in legend to Fig. 1. uptake of 1*&I-glucagon. Plasma membranes (25 rg of protein) Fro. 3 (center). EfIocm of GTP on dissociation of bound r*SI- were incubated in 0.125 ml of medium containing 2.5yo albumin, glucagon from plasma membrnnes in the presence and absence 5.2 nM `2LI-glucagon (0.4 X 10' cpm prr pmolc), Tris-HCI, p1-I 7.6, of unlabeled glucagon. Kxperiments were conducted in the same and the indicated concentrations of GTP. Incubation time was manuer and under the identiral incubation conditions described 15 min at 30". The quantity of glucagon bound to plasma mem- for the first stage of incubation in Fig. 1. In the second stage, branes was determined from the amount of radioactivity hound and the specific activity of the lnbeled glucagon, as described incubations were continued at 30" in the absence and presence of previously in detail (1). GLUCAGON (Ml MINUlES Fro. 5 (I&). Effects of GTP on the uptake of varying concen- trations of i*SI-glucagon by plasma membranes. Plasma mem- branes (25 pg of protein) were incubated for 20 min at 30" in 0.125 ml of medium containing 2.5% albumin, I rnM EDTA, 20 mre Tris-HCl, pH 7.6, and the indicated concentrations of l*61-gluca- gon (320,000 cpm per pmole). GTP, when present, was 4 PM. The method of separating bound from free labeled hormone and calculations of amount of glucagon bound per mg of membrane protein are described in Reference 1. Fro. 6 (right). Effects of GTP on the time course of binding of glucagon in the presence and absence of EDTA. Plasma mem- branes (25 pg of protein) were incubated at 30" in 0.125 ml of medium containing 2.57, albumin, 5.4 no i~rI.glucsgon (320,000 cpm per pmole), and Tris-HCl, pH 7.6. When present, the con- centrations of EDTA and GTP were 1 n,~ and 1 FM, respectively. The method of separating bound from free labeled hormone and calculations of amount of glucagon bound per mg of protein are described in Reference 1. IO-6 ro-5 m-4 to-3 NUCLEOTIDE ADDED It'!) FIG. 7. Effects of various concentrations of GTP, GDP, and GMP-PCP on dissociation of bound labeled glucagon from plasma membranes. Experiments were carried out in the absence of EDTA but otherwise under the same manner and under the in- cubation conditions described in legend to Fig. 1 with the excep- tion that either GTP, GDP, or GMP-PCP were added simul- taneously with unlabeled glucagon (5 PM) during the second stage of incubation. tidases (7). Since EDTA had a permissive effect on the action of low concentrations of GTP, it was suspected that the chelator may prevent the breakdown of GTP by removing metal ions bound to the membranes. We examined this possibility by measuring the hydrolysis of G'l'P-y-82P, 20 pM, in the same me- dium used for the binding studies and with the same concentra- tion of plasma membranes (200 fig of protein per ml). In the absence of EDTA, GTP was hydrolyzed at a rate of 14 nmoles per min per nig of protein. EDTA, at I mlr, inhibited the hydrolysis of GTP by 96%,, suggesting that its permissive effect on the actions of GTI' may be due to inhibition of nucleotidases present in liver membranes. In studies that will be reported later in detail, it has been found that 5 mbf Mg++ or Ca'+ dou- bled the rate of hydrolysis of G'I'P. TAIILE .I\' Eflecls of GTP on dissociation of botrnd Inhelcd qlumgon from liar tncmbrancs inccibdd nl 0" ant1 SO" Liver membranes (0.2 mg per ml) were incubated in medium containing 2.5ye albumin, 20 mM Tris-HCl, pH 76, and 4.5 IIM 1*61-glucagon for 15 min at 30". IIalf of the suspension was placed in an ice bath and incubated at 0" for 15 min in presence of 5 PM unlabeled glucagon with or without GTP (1.0 PM). The other half was incubated with the same additions but at 3(i". The per- centage of bound labeled glucagon dissociated during the second stage of incubation was determined as described previously (1). Dissociation of bound labeled glucagon Incubation temperature -GTP I + GTP % 0" 4 I 46 30 9 49 TABLE V Effects of CMB on upiake and disson'ation of labeled glucagon in liver membranes and on efects of GTP on these processes Plasma membranes (0.2 mg per ml) were incubated in medium containing 2.5y0 albumin, 1 mM EDTA, 20 mre Tris-HCl, pH 7.6, 4.5 nM l*D1-glucagon. When present, CMB and GTP were 0.2 mM and 1.0 FM, respectively, and were added at izo time in uptake studies and at the moment of addition of unlabeled glucagon in studies of dissociat.ion. Incubations were for 15 min at 36" during both types of studies which were carried out as described in the legend to Table II. Cb.fB + uptake of glucagon Bound `=I-glucsgon diisociatcd -GTP +GTP -GTP +GTP pnrolcr/ng pro1ein % 1.18 j 0.63 0.46 j 0.34 ii 1 ii: Conrporative `Eflecta of 5'-Gucnylyl Melhykne Diphosphafe, GTP, and GDP on Dissociation of Bound Gluccsgon-The effects of GMP-PCPi (an analogue of GTP containing a carbon atom in place of oxygen between the /3 and y phosphate groups) were compared with those of GDP and GTP on dissociation of bound glucagon. GMP-PCP can only be hydrolyzed to give 5'-GMP (S), which is inactive on glucagon binding. As shown in Fig. 7, GMP-PCP also stimulated dissociation of bound glucagon nl- though at concentrations 100 times that of either CTP or GDP which had essentially equivalent actions at all concentrations. Other Chmacteristics of GTP &ions 012. Binding of Glucagon- The effects of GTP on dissociation of bound labeied glucagon were equivalent at either 0" or 30" of incubation as illustrated in Table IV. In the absence of GTP, dissociation was slight at either temperature (less at O", see also Reference 1). It should be noted that EDTA, which stimulates dissociation of bound labeled glucagon (l), was omitted in these experiments. Shown in Table V are the effects of the sulfhydryl reagent, CRIB, at 0.2 rnq on both uptake of gluc:rgon and dissociation of bound labeled plucagon, and on the actions of GTP on these processes. CMB inhibited binding of gluargon bg 61 `;;, and either abolished or inhibited markedly the effects of GTP on * The abbreviations used are: GMP-PCP, 5'guanylyl mcthylene diphosphonate; CMB, p-chloromercurihenzoaie. 1876 Glucagon-sensitive Adenyl Cyclase. IV Vol. 246, No. 6 either upt:lko of gluc:~go~~ or di~soci:ttion of bound labeled hor- mane . In stud& not recorded, it W:W fomld that sodium fluoride (10 mM), in the presence or :~hsrnce of ,1Ig+f (5 rnh%) or of EIWA (1 mM), did not alter either the proccsscs of binding (uptake or dissociation of bound 1:&&d hormone) or the actions of GTP on these processes. The previous studies (1, 3) showed that agents such as phos- pholipase A, urea, and detergent* diminished both the binding of glucagon and activation of ndenyl cyclase by the hormone. While these correlations help to establish the relationship be- tween binding sites and the aden)- cyclase system and provide sr)ne insight into the chemical and physical properties of the bin:ling sites, the destructive actions of these agents on both proc- eases do not permit the conclusion that the binding process is necessarily or uniquely involved in the activation of adenyl cyclnse by glucagon. For this aspect of the problem, more meaningful correlations can be derived from studies with agents that stimulate or alter both processes in a nondestructive manner. Two phenomena observed in this series of studies seem to fit this criterion. One is the stimulatory effect of EDTA on both activation of adenyl cyclase by glucagon (2) and dissociation of bound glucagon (1). The other, reported in this study, is the action of GTP or GDP, at concentrations approaching that at which glumgon binds or activates adenyl cyclase in liver mem- branes, on the binding sites for glucagon. The following study will show that guanyl nucleotides stimulate the response of adenyl cyclase to glucagon (9). The mechanism by which EDTA or the guangl nucleotides alter binding or activation of adenyl cyclase remains unknown, but it can be suggested, in the case of EDTX, that membrane-bound metal ions may be involved in both processes. The actions of the guanyl nucleotides appear to differ both qualitatively and quantitatively from that of EDTA on the binding process. GTP and GDP, equally and at concentrations s 10~ as 0.05 ~11, stimulated both the rate and degree of dissociation of bound glucagon, decreased the uptake of submasimal concentrations of glucagon, and decreased the apparent affinity of the binding sites for glucagon. The relatively slow method of measuring binding in these studies did not permit assessment of the effects of the uucleotides on the initial rates of association or dissociation of the hormone at its binding sites. It can be stated, however, that the nucleotides induce a change in the state or properties of the bind- ing sites leading to a more rapid attainment of constant, although lower levels of bound glucagon and to a change in the apparent affinity of the binding sites for glucagon. hlthough the mechanism by which the guanyl nucleot,ides alter the state of the glucagon binding sites remains mlknown, the following observations suggest that they act through binding and not by phosphorylation of the material that binds gluragon. Both GTP and GDP were equally effective at the same concen- trations, indicating that interconversion of one nucleotide to the other is not. required for their effects. Thcrc was no requirement for a divnlent ion as might be expected for a phosphorylating meclu\uism. Intlced, 13DT.1 sustained the effects of GTP on binding by inhibiting the breakdown of the nucleotide by 31g'+- or Cn++-requiring nucleotitlases present in the membranes. (:;\Il'-Xl', a llon1)llosl)hor~l:ltiI~g analogue of GTY, mimicked the actions of G'l'l' or C: Dl', although at concentrations 100 times `hat of the natural c~u~~pou~~ds. The lower apparent affinity of the aualogue for the glucxgon binding sitrs may be related to the recent report (10) that the bond angles of the tliphosphonate bond (P-C-P) are significantly different from those of the pyrophosphnte bond (P-&~I'). It is also possible that the analogue escrts its action by inhibiting the breakdown of endoge- IlOUS, membrane-bound guanyl nucleotides. Finally, GTP stimulated dissociation of bound labeled glucagon equally at. 0" and 30", suggesting that the actions of GTP are related to a bind- ing rather than an enzymatic process. In this regard, the find- ing that cyclic 3',5'-GMP and 5'-GRIP did not alter binding of glucagon rules out the possibility that nucleotidases or guanyl cyclase, the latter reported to be present in particulate fractions of rat liver (ll), may be responsible for the actions of GTP or GDP. At low concentrations, GDP and GTP did not show comgeti- tive intelaction with glucagon at the binding sites. This ws evident from the finding that the nucleotides act at 0.05 ,.N even in the presence of 5 pM glucagon in the incubation medium. It would appear, therefore, that the nucleotides exert an allosteric type of action on the glucagon binding sites. The effects of GTP were reversible and concentration dependent, suggesting that breakdown by nucleotidases or other factors that influence metabolism or binding of guanyl nucleotides may regulate the actions of the nude&ides on the glucagon-binding process. Both binding of glucagon and the actions of GTP on binding were in- hibited by a sulfhydryl reagent, which raises hhe possibility that regulation of both processes could be affected through modifica- tion of sulfhydryl groups. It was also found in this study that ATP, ADP, and the pyrimi- dine nucleotides, UTP and CTP, also affect,ed glucagon binding in the .same manner as GTP or GDP although at concentrations at least four orders of magnitude higher than the minimal effee- tive concentration of the guanyl nucleotides. It is possible that the effects of the other nucleotides are due to contaminated guanyl nucleotides. In any case, the consequence of the effects of the adenine nucleotides on glucagon binding is that studies of the specific effects of guanyl nucleotides on the response of adenyl cyclase to glucagon necessitates the use of low concentrations (less than 0.5 mM> of ATP, the substrate for adenyl cyclase. The following paper (9) examines the question of whether the effects of the guanyl nucleotides on the binding of glucagon bear relationship to the actions of glucagon on the adenyl cyclaae system in rat liver plasma membranes. REFERENCES 1. RODBELL,M.,RRANS, H.M.J., PoHL,S. L., AND BIRXUAGMEH, L., J. Biol. Chem., 246, 1861 (1971). 2, POHL, S. L., BIRNBAUMEH, L., AND RODBCLL, hf., .I. Biol. Chem., 246, 1849 (1971). 3. BIRNBAIJMER, L., POHL, S. L., AND HODRELL, M., J. Biol. Chem., 246, 1857 (1971). 4. SCGINO, Y., AND MIYOSHI, Y., J. Eiol. Chem., 239, 2360 (1964). 5. BRAN, G. A., linal. B&hem., 1, 279 (IPGO). 6. Lo\\'I~Y,(~. H.,ROSERHOL-GH,N.J., FARR, A.L., AND I:.\suA~r>, 1~. J., J. Biol. Chem., 19S, 265 (1951). 7. EMMELOT. P.. Bos. C. J.. BRNEDETTI, E. I,.. AND ltcal~~. P. H., &o&m. biophys: Acla, 90, 126 (1964j. 8. HERSHICY, J. W. B., AND MONIK), It. E., J. dfol. B?.ol., 18, 68 (1'9G8). 9. ROI)IIEI,L, M., BIRNII.\UYER, I,., Pear,, 8. I`., .~NI) l<~c.\sr, Il. M. J., J. Biol. 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