Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues. Detection of vicinal diols by mass fragmentography

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

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.     

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.     

Alterations in cerebral β-adrenergic receptor-adenylate cyclase system induced by halothane,ketamine and ethanol

The effect of halothane, ketamine and ethanol on β-adrenergic receptor adenylate cyclase system was studied in the brain of rats. An anesthetic concentration of halothane and ketamine added in vitro decreased the stimulatory effect of norepinephrine on cyclic AMP formation in slices from the cerebral cortex. On the other hand, ethanol increased the basal activity of cerebral adenylate cyclase without affecting on the norepinephrine-stimulated activity. The increase of the basal activity induced by ethanol was not antagonized by propranolol, a β-adrenergic antagonist. In the crude synaptosomal (P₂) fraction, these drugs had no significant effect on the basal adenylate cyclase activity, binding of [³H]dihydroalprenolol to β-receptor, and binding of [³H]guanylylimido diphosphate ([³H]Gpp(NH)p) to guanyl nucleotide binding site. In contrast, the adenylate cyclase activity stimulated by Gpp(NH)p or NaF was significantly inhibited by an anesthetic concentration of these drugs. An anesthetic concentration of these drugs increased the membrane fluidity of P₂ fraction monitored by the fluorescence polarization technique. The addition of linoleic acid (more than 500 μM) also induced not only the increase of fluidity, but also the decrease of Gpp(NH)p- or NaF-stimulated adenylate cyclase activity in the cerebral P₂ fraction. The present results suggest that general anesthetics may interfere with the guanyl nucleotide binding regulatory protein-mediated activation of cerebral adenylate cyclase by disturbing the lipid region of synaptic membrane.     

Stimulation of mammalian adenylate cyclase activity with guanosine 5′-tetraphosphate and other guanine nucleotides

Guanosine 5′-tetraphosphate (GTP₄) stimulated mammalian adenylate cyclase activity at concentrations down to 1 μM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP₄ stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3′, 5′-monophosphate and 5′-guanylylimidodiphosphate (GMP · PNP) also stimulated mammalian adenylate cyclase activity. GMP · PNP irreversibly activated, whereas GTP₄ and GTP reversibly activated adenylate cyclase. Adenosine 5′-tetraphosphate (ATP₄) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP₄. The formation of cyclic AMP by GTP₄-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H⁺), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP₄ was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The β-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP₄, thus, suggesting that GTP₄ and β-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP₄ or GMP. PNP, thus indicating that GTP₄ resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to β-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2′-monophosphate, guanosine 3′-monophosphate or guanosine 2′,5′-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.     

The role of GTP in prostaglandin E1 stimulation of adenylate cyclase in platelet membranes.

Adenylate cyclase activity in platelet membrane preparations was measured in the presence of prostaglandin E1 (PGE1), GTP and a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). A dose-dependent inhibition of adenylate cyclase by GDP[beta S] was observed that could be reversed either by adding increased amounts of GTP or of PGE1.     

Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.     

Influence of cholera toxin on the regulation of adenylate cyclase by GTP.

In the presence of NAD⁺, cholera toxin activates adenylate cyclase in membranes of S49 mouse lymphoma cells. The following evidence supports the hypothesis that the toxin acts by inhibiting a specific GTPase associated with a guanyl nucleotide regulatory component of hormone-responsive cyclase: 1. GTP alone markedly stimulates cyclase activity in toxin-treated, but not in untreated membranes; 2. The poorly hydrolyzable GTP analog, guanosine 5′-(β,γ-imino) triphosphate (Gpp(NH)p), stimulates cyclase equally well in toxin-treated and untreated membranes; 3. Cyclase activation by isoproterenol plus GTP persists in toxin-treated membranes, but not in controls, after addition of propranolol; 4. GTP is a more potent competitive inhibitor of the irreversible activation of cyclase by Gpp(NH)p in toxin-treated than in untreated membranes.     

Dopamine sensitive adenylate cyclase in planaria Dugesia gonocephala

1. The adenylate cyclase activity present in the particulate fraction of planaria homogenates has been characterized.2. The enzyme requires divalent cations (Mg²⁺), and a K_m for ATP of 0.58 at 30°C was measured.3. GTP and Gpp(NH)p, in an optimal range of 10⁻⁴–10⁻⁵M, increase the enzymatic activity.4. In the presence of GTP, dopamine stimulates the adenylate cyclase and its action is inhibited by dopaminergic antagonist.5. Both D-1 and D-2 selective dopaminergic agonists stimulate the enzymatic activity and their action is selectively antagonized by D-1 and D-2 antagonists.6. The high concentrations required for some D-1 and D-2 agents to be effective, suggest an only partial consistency with mammalian dopaminergic receptors.     

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membranes was measured, and compared with the effect of PGE1. When GTP was added in concentrations up to 10 microM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S] ) resulted in a dose-dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.     

Activation of rat liver adenylate cyclase by guanosine 5''-[beta,gamma-imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein.

The effects of guanosine 5'-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi-irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state.     

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.     

A kinetic analysis of activation of smooth muscle adenylate cyclase by forskolin

Forskolin action was studied using uterine smooth muscle adenylate cyclase, an enzyme form that is slowly and irreversibly activated by treatment with nonhydrolyzable GTP analogs. Activation of the particulate smooth muscle enzyme by prolonged treatment with Gpp[NH]p (guanyl-5′-yl imidodiphosphate) at 24 °C followed simple Michaelis-Menten kinetics with respect to the guanine nucleotide. Under these treatment conditions, forskolin increased both the V_max and the K_m for Gpp[NH]p, suggesting diterpene action affected the guanine nucleotide-binding coupling factor. Sensitivity of a detergent-solubilized form of the enzyme to stimulation by both Gpp[NH]p and forskolin was much more labile at 4 °C than was the Mn⁺² sensitivity of the catalytic subunit. In the particulate form, the catalytic subunit was more resistant to the denaturing effects of N-ethylmaleimide than was its sensitivity to stimulation by Gpp[NH]p or forskolin. Forskolin stimulation of the particulate form of the enzyme followed simple Michaelis-Menten kinetics with respect to the concentration of the diterpene. Denaturation of the enzyme by treatment with N-ethylmaleimide lowered the V_max and increased the K_m for forskolin, further suggesting that forskolin had an indirect effect on the activity of the catalytic subunit. These results could be accounted for if the diterpene, like Gpp[NH]p, was bound by the coupling factor.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

Differential Sensitivity of Basal and Opioid-Stimulated Low Km GTPase to Guanine Nucleotide Analogs

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.