Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.     

Effects of pertussis toxin-catalyzed ADP-ribosylation on interactions of transducin and the inhibitory GTP-binding protein of adenylate cyclase with guanyl nucleotides

Release of bound [3H]Gpp(NH)p from NG108-15 cell membranes was induced by carbamylcholine, enkephalinamide, and norepinephrine, all of which inhibit adenylate cyclase. Release was blocked by antagonist, was greater with multiple agonists than with one, and required guanyl nucleotides. With membranes from pertussis toxin-treated cells, both total [3H] Gpp(NH)p binding and agonist-induced [3H]Gpp(NH)p release was decreased. ADP-ribosylation by toxin of transducin, the retinal GTP-binding protein which is similar in structure and function to that in cyclase, decreased [3H]Gpp(NH)p binding. Thus, the inability to demonstrate agonist-induced [3H]Gpp(NH)p release from toxin-treated NG108-15 membranes may result in part from absence of bound [3H]Gpp(NH)p.     

Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.     

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.     

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca²⁺-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μm GTP and 3.5-fold by 100 μm Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca²⁺ concentrations; both phases of this response occurred at lower free Ca²⁺ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

Irreversible activation of adenylate cyclase of toad erythrocyte plasma membrane by 5′-guanylylimidodiphosphate

Summary The irreversible activation of adenylate cyclase by 5 guanylylimidodiphosphate, a phosphoramidate analog of 5GTP, has been examined in toad (Bufus marinus) plasma membranes using the technique of preincubating the membranes with the nucleotide under various controlled conditions followed by washing and subsequent assay of enzyme activity. Activation of adenylate cyclase by Gpp(NH)p, but not GTP, is essentially permanent and persists following extensive washing, prolonged incubation at 30°C in the absence of the nucleotide, and after dissolution of the membranes with Lubrol PX. (–)-Isoproterenol increases the activation observed with maximal concentrations of Gpp(NH)p from eight- to 10-fold (in the absence of hormone) to 50- to 100-fold; final activities as high as 10–15 nmoles of cyclic AMP per min per mg protein are achieved. The activated state obtained with isoproterenol and Gpp(NH)p is also permanent and is not inhibited by propranolol. The synergism between Gpp(NH)p and hormone requires the simultaneous presence of these compounds, and the time-dependent enhancement of activation with (–)-isoproterenol may be interrupted by addition of propranolol.The stimulation is slow, and may proceed for as long as 45 min at 30°C in the presence of maximal concentrations of Gpp(NH)p and (–)-isoproterenol. Very little activation occurs at 0°C. The time course of activation at 30°C exhibits an accelerating phase lasting from 5 to 30 min when Gpp(NH)p is added directly during assay of cyclase activity or when the membranes are preincubated for various times and washed prior to assay for a fixed time. The lag period occurs in the presence and absence of (–)-isoproterenol, although the rate of increase in velocity is greater with hormone. The length of the accelerating phase decreases with increasing concentrations of Gpp(NH)p, although it is still evident with maximal levels of Gpp(NH)p and hormone. However, prewarming the membranes at 30°C for 10 min in the absence of Gpp(NH)p or (–)-isoproterenol results in an immediate onset of linear activation at a rate which is achieved in untreated membranes only after about 10 min. The events occurring during prewarming at 30°C are readily reversible since chilling the warmed membranes to 0°C results in a time course of activation identical to that of membranes maintained at 0°C until addition of Gpp(NH)p.Activation is proportional to the concentration of Gpp(NH)p within the range of 10^–8 to 10^–4 mm. The apparent affinity for Gpp(NH)p increases with increasing time of incubation. The primary effect of increasing the concentration of Gpp(NH)p is to decrease the time required to obtain a maximal rate of activation.The possible relevance of these findings to the mechanism of action of Gpp(NH)p, adenylate cyclase and hormones is discussed within the context of current views of biological membranes which recognize the lateral mobility of membrane molecules.     

The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortened by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited foreskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

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.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mode of action of chloride on rabbit heart adenylate cyclase

The mechanism by which chloride stimulates adenylate cyclase was investigated. Depletion of GDP increased basal adenylate cyclase activity and reduced the stimulation by isoprenaline. Restoration of bound GDP partially reversed these effects. Chloride stimulated cyclase activity by the same proportion in control, GDP-depleted and GDP-restored preparations, as did Gpp(NH)p. Fluoride increased adenylate cyclase activity to the same final level in both GDP-depleted and GDP-restored membranes; addition of Gpp(NH)p as well as fluoride had no further effect. Solubilisation of adenylate cyclase reduced the stimulatory effect of Gpp(NH)p only slightly, but greatly attenuated the activation by chloride. We conclude that chloride does not stimulate cyclase activity by an action on GDP exchange. Activation by chloride may be due to a disrupting or chaotropic effect on membrane/protein interactions.     

Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

The regulation of adenylate cyclase activity in turkey erythrocyte membranes by forskolin

The mechanisms by which forskolin stimulates adenylate cyclase activity in turkey erythrocyte membranes and is influenced by manganese and Gpp(NH)p were studied. Forskolin-dependent adenylate cyclase activity in particulate turkey erythrocyte membranes is enhanced following preincubation of membranes with isoproterenol and GMP (cleared membranes). In contrast, solubilization of turkey erythrocyte membranes, previously cleared, renders them relatively refractory to forskolin but not to Gpp(NH)p. Whereas adenylate cyclase activity due to the simultaneous presence of forskolin and Mn2+ in particulate turkey erythrocyte membranes is additive, their copresence becomes synergistic after solubilization. The apparent Kact for forskolin activation of adenylate cyclase is not influenced by clearance or by the presence of Mn2+ in particulate turkey erythrocyte membranes. Following solubilization, the Vmax for forskolin-dependent adenylate cyclase activation determined in the presence of Mn2+ is also independent of clearance. Forskolin activation of turkey erythrocyte adenylate cyclase appears to be influenced at sites in addition to the catalytic unit.     

Characteristics of 5'-guanylyl imidodiphosphate-activated adenylate cyclase.

Characteristics of adenylate cyclase stimulation by the GTP analog 5'-guanyl imidodiphosphate Gpp(NH)p have been examined in intact frog erythrocytes, frog erythrocyte membranes, and solubilized canine myocardial preparations. Gpp(NH)p caused marked enzyme activation in the erythrocyte membranes and in solubilized myocardial preparations, but had much lesser effects in intact cells. Enzyme activation by Gpp(NH)p exhibited a definite lag period, requiring 10 to 15 min for complete activation at 37 degrees. Activation was essentially irreversible after a 5-hour dialysis sufficient to reduce the Gpp(NH)p levels below threshold for stimulation. Gpp(NH)p-"activated" enzyme differed from native enzyme in several respects, such as its greater temperature stability, and its insensitivity to further stimulation by other activators, such as catecholamine or fluoride. These differences suggest that the enzyme, once fully activated by Gpp(NH)p, may have undergone some modification that is not subject ot facile reversal.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

A simple test for enhanced guanyl nucleotide exchange in brain adenylate cyclase systems activated by neurotransmitters

The mechanism of receptor-induced activation of adenylate cyclase has been proposed to involve an enhanced exchange of GDP for GTP. The kinetics of this process have not been investigated so far in the brain due to a spontaneous activation of the enzyme by guanyl nucleotides, which precludes the ability to follow receptor-dependent events. We show that it is possible to investigate the mechanism of receptor action in such systems by using a combination of guanosine 5'-(beta-gamma-imino)triphosphate (Gpp(NH)p) and guanosine 5'-(2-O-thio)diphosphate (GDP beta S). In pineal membranes, beta-adrenergic agonists increase the rate of adenylate cyclase activation by 10 or 100 microM Gpp(NH)p about 40-fold (0.023-0.9 min-1 kact) and decrease the inhibitory potency of GDP beta S nearly 1000-fold. As a result, 100 microM GDP beta S which blocks 90% of the activation by 10 microM Gpp(NH)p has no inhibitory effect in the presence of 10 microM Gpp(NH)p and 10 microM noradrenaline or isoproterenol. In caudate nucleus, dopamine does not appear to increase the rate of activation of adenylate cyclase by 10 microM Gpp(NH)p. Nevertheless, 100 microM GDP beta S blocks 90% of the activation by 10 microM Gpp(NH)p but has no inhibitory effects in the presence of dopamine. Thus, one can demonstrate that even weakly activating receptors have the capacity to facilitate a functional exchange of GDP beta S for Gpp(NH)p and measure the efficacy of the interaction between the receptor and the functionally linked guanyl nucleotide subunit.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.