Regulation of glucagon receptor binding. Lack of effect of Mg and preferential role for GDP

The effects of Mg2+ and guanine nucleotides on glucagon binding to its receptor were studied using [125I-Tyr10]monoiodoglucagon. Contrary to findings with beta-adrenergic receptors, high affinity binding of the stimulatory hormone was not dependent on Mg2+ and low affinity binding could be obtained on nucleotide addition regardless of presence of Mg2+. GDP, guanyl-5'-yl thiophosphate (GDP beta S), GTP, and guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) were all able to induce low affinity hormone binding. Since the Ns component of adenylyl cyclase, with which the receptor interacts, is inactive in stimulating the catalytic component C of adenylyl cyclase in the absence of Mg2+, both before and after GDP addition, it is suggested that Ns has at least two domains that change conformation independently of each other: a r domain, that interacts with the receptor and confers to it high affinity binding, and a c domain, that interacts with the catalyst C and stimulates it. It is suggested further that Ns is r+c- when stabilizing the receptor in its conformation with high affinity for hormone, and r-c- when under the influence of GDP which results in the receptor adopting the conformation that exhibits low affinity for the hormone. Comparison of potencies of the four nucleotides to induce low affinity binding showed that GDP and GDP beta S were equipotent and 10 times more potent than GTP and 100 times more potent than GMP-P(NH)P. Under the conditions used it was impossible to substantiate that the effects of GTP or GMP-P(NH)P were not due to formation of GDP from GTP or presence of GDP-like material in GMP-P(NH)P. It is suggested that, contrary to widely held opinions, GDP and GDP-like compounds, and not GTP or its analogs, are responsible for the lowering of the affinity of adenylyl cyclase stimulating receptors for their hormones or agonists. Furthermore, the experiments suggest that the c+ conformation of the c domain of Ns co-exists with the r+ and not the r- conformation of its r domain.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Photolysis of water coupled to nitrate reduction by Nostoc muscorum subcellular particles.

The binding of tritiated guanylylimidodiphosphate ([³H]GMP-P(NH)P) to turkey erythrocyte ghosts was studied in parallel with the activation by GMP-P(NH)P of adenylate cyclase. The high affinity binding capacity for GMP-P(NH)P, 50 pmoles per mg protein, exceeds the estimated quantity of adenylate cyclase of 1 pmole per mg of protein. The rate of nucleotide binding is not affected by isoproterenol. Further, in the presence of the hormone the rate of binding is much slower than the rate of activation. Although the rate of dissociation of bound [³H]GMP-P(NH)P is negligible at 37°, it is increased dramatically by unlabeled GMP-P(NH)P, GTP, EDTA, ATP, AMP-P(CH₂)P, or p-aminophenylmercuric acetate. In contrast, the rate of decay of the GMP-P(NH)P-simulated state is not altered by these agents. Thus, the major fraction of GMP-P(NH)P binding to membranes is not relevant to cyclase activation.     

Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

Kinetic studies on rat liver and beef heart mitochondrial ATPase. Evidence for nucleotide binding at separate regulatory and catalytic sites.

Mitochondrial ATPases from rat liver and beef heart were used to study the effects of guanylylimidodiphosphate (GMP-P(NH)P) and adenylylimidodiphosphate (AMP-P(NH)P) on the kinetics of MgATP, MgITP, and MgGTP hydrolysis. AMP-P(NH)P was a noncompetitive inhibitor of hydrolysis of all substrates with the rat liver enzyme, whether activating anions were present or not. Also with the liver enzyme, AMP-P(NH)P caused only MgATP hydrolysis to appear to have positive cooperativity. With the beef heart enzyme, AMP-P(NH)P was a competitive inhibitor of ATPase activity and caused positive cooperativity; it gave noncompetitive patterns with GTP or ITP as substrates. In both enzyme systems, GMP-P(NH)P gave complex inhibition patterns with MgATP as the substrate, but was a competitive inhibitor of MgITP and MgGTP hydrolysis. These results are interpreted as indicating the existence of two types of nucleotide binding sites, with varying degrees of specificity and interaction on the ATPase molecules from both sources. It is postulated that MgATP and AMP-P(NH)P bind to regulatory site while MgATP, MgGTP, Mgitp, and GMP-P(NH)P bind to the catalytic site.     

Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.     

The molecular size of adenylate cyclase in the absence and presence of nucleotide and hormone effectors

The molecular size of adenylate cyclase solubilized from frog erythrocyte membranes by digitonin extraction has been determined by chromatography on Sepharose 6B. Regardless of whether the membranes are exposed to catecholamines, GPP(NH)P, NaF or no effector prior to solubilization, the apparent molecular size of the adenylate cyclase enzyme is the same. Furthermore, a similar elution profile for the enzyme is observed when the catalytic activity in the eluates is measured in the presence of Mn++, rather than Mg++. Since it is generally assumed that the persistent activation of adenylate cyclase by GPP(NH)P requires interaction of the catalytic moiety with the guanine nucleotide regulatory site, it appears that the adenylate cyclase activity detected in the column eluates represents an intact catalytic-regulatory site complex. The adenylate cyclase activity derived from catecholamine pretreated frog erythrocyte membranes does not co-elute with catecholamine-occupied beta-adrenergic receptors, indicating that the agonist-promoted increase in apparent receptor size observed here and in earlier studies does not represent a physical coupling of the receptor and the adenylate cyclase enzyme.     

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.     

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.     

Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

Calmodulin-Sensitive and Calmodulin-Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides

The interaction between the Ca2+-binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal particulate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5'-(beta,gamma-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 microM GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The calmodulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

Ontogenetic study of the regulatory and coupling function of guanyl nucleotide-binding proteins in the hormone-sensitive adenylate cyclase system

Reconstitution of catecholamine-sensitive adenylate cyclase from chick embryonic muscle membranes and guanyl nucleotide-binding proteins of mature rabbit muscle makes it possible to reveal the coupling (potentiating) effect of these nucleotides 1 week earlier than in the native condition. The effective insertion of guanyl-nucleotide-binding proteins into the embryonic membrane coincides with the onset of a pronounced increase in membrane lipid fluidity during the course of embryogenesis. The different ontogenetic time-courses for the origination of the two guanyl nucleotide effects, on catalytic adenylate cyclase activity (in early embryogenesis) and on the coupling process (in postembryonic life), suggest the existence in this system of two separate guanyl-nucleotide-binding proteins performing regulatory and coupling functions, respectively.     

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.     

On the role of guanyl nucleotides in adenylate cyclase system of embryonic skeletal muscle

The effects of guanyl nucleotides on the catalytic and catecholamine-stimulated activities of adenylate cyclase in developing chick skeletal muscles were studied. GTP and guanylyl imidodiphosphate stimulate the cyclase catalytic activity already at the early embryonic stages without having potentiating influence on the catecholamine-stimulated activity in embryonic muscle. In a distinct and regular form this effect can be observed only after hatching. Therefore during embryogenesis the coupling function of the GTP-binding component of adenylate cyclase system characteristic of its mature state is not manifested. The effects of the nucleotide suggest that they occur as two independent processes.