Cytochemical localization of K+-dependent p-nitrophenyl phosphatase and adenylate cyclase by using one-step method in human washed platelets

Summary Ultrastructural cytochemical localization of ouabain-sensitive, potassium dependent p-nitrophenyl phosphatase (K⁺-NPPase) of the Na⁺-/K⁺-ATPase complex and adenylate cyclase (cAMPase) activities, in washed inactivated human platelets, are described. The one-step lead-citrate method, under similar incubation conditions, was used to determine both activities. K⁺-NPPase appeared in both plasma membrane and the surface-connected canalicular system (SCCS) of the platelets. These data suggest a uniform distribution of the enzyme throughout membrane systems which are in contact with the external medium. cAMPase activity was strictly localized in tubules of the dense tubular system (DTS) when incubation medium contained prostaglandin E₁, prostaglandin D₂ or forskolin, at concentrations known to stimulate the enzyme in platelets that are intact. This fact and the inhibition of cytochemical reaction by thrombin confirm that the one-step lead-citrate method is a useful procedure in determining adenylate cyclase, abolishing the unfavorable conditions of previously reported methods.     

Cytochemical localization of adenylate cyclase in the dense tubule system of human blood platelets stimulated by forskolin, prostacyclin and prostaglandin D2

Platelets were briefly fixed in paraformaldehyde/glutaraldehyde and then incubated with 5'-adenylyl imidodiphosphate under conditions suitable for the cytochemical detection of adenylate cyclase activity. The adenylate cyclase activity of these platelets retains the ability to respond to prostaglandins E1, D2, I2 (prostacyclin), forskolin and fluoride. Sites of stimulated adenylate cyclase activity were localized cytochemically by the reaction of lead with the reaction product imidodiphosphate to form deposits of lead imidodiphosphate that are visible in the electron microscope. Reaction product deposition was seen only in the dense tubule system of human platelets when the incubation medium contained forskolin, prostacyclin, or prostaglandin D2 at concentrations known to stimulate the enzyme in intact platelets. Epinephrine, an antagonist of adenylate cyclase inhibited the cytochemical reaction stimulated by prostacyclin. The fact that the cytochemical reaction was induced by agonists that stimulate the enzyme through two different types of prostaglandin receptors and by forskolin, which acts distal to the receptors, confirms that the method specifically detects adenylate cyclase. The presence of adenylate cyclase in the dense tubules may be significant for the regulation of intracellular Ca2+ and arachidonic acid metabolism by this membrane system.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

Summary The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin-or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E₁ plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.     

Desensitization of prostaglandin-activated platelet adenylate cyclase.

Prostaglandin D2 (PGD2) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD2 washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 x 10(-5) M PGD2 for 2 hr resulted in a 45% decrease in PGD2 activation of adenylate cyclase and a 25% decrease in stimulation by PGE1. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD2. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10(-7) M PGD2. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD2 and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.     

Ultracytochemical study of adenylate cyclase localization in the adenohypophysis of rats

Using an electron cytochemical method and adenylylimidodiphosphate (AMP--PNP) as substrate, the localization of adenylate cyclase activity was determined in the rat's adenohypophysis. This activity was discovered in the perinuclear space, in the canaliculi of the endoplasmic reticulum and Golgi complex, in mitochondria, on the external surface of the plasma membrane. In sinusoidal capillaries, the reaction product was localized on the plasma membrane, in perinuclear space, endoplasmic reticulum and mitochondria. The addition of isoproterenol and sodium fluoride to the incubation medium led to a rise in adenylate cyclase activity.     

Schizophrenia and reduced cyclic AMP production: evidence for the role of receptor-linked events

Reduced cyclic AMP (cAMP) production has been found in platelets of schizophrenic patients. cAMP is generated physiologically as a result of a series of steps beginning with receptor activation by a ligand, progressing through activation of the enzyme protein, adenylate cyclase. The deficit of cAMP found in the schizophrenic population may occur at any one, or at multiple steps in this cascade. The present study attempts to discriminate whether impaired adenylate cyclase itself was responsible for the cAMP deficit or whether abnormalities in receptor events or linkage are present in schizophrenics. The production of cAMP following direct stimulation of adenylate cyclase by NaF was contrasted with receptor mediated activation of adenylate cyclase by prostaglandin E1 (PGE1) in disrupted platelet preparations from schizophrenics and normal controls. cAMP formation stimulated by NaF was not different in platelets of schizophrenics as compared to controls, however, platelets of schizophrenics showed reduced response to PGE1 stimulation. The authors interpret these findings as evidence for a membrane associated abnormality of either receptor or receptor-adenylate cyclase linkage in the schizophrenias.     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Multiple effects of guanine nucleotides on human platelet adenylated cyclase.

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occurring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Selective effects of gold (III) on parathyroid hormone-, prostaglandin E2- and guanine nucleotide-sensitive adenylate cyclase

Gold(III) (Au(III)) up to 0.25 microM increased parathyroid hormone- and prostaglandin E2-sensitive chick osteoblast adenylate cyclase activity without affecting 5'-guanylylimidodiphosphate-stimulated enzyme activity. Au(III) at 5-50 microM inhibited hormone- and nucleotide-mediated activation of adenylate cyclase. Basal adenylate cyclase activity was not influenced by Au(III) in the given concentrations. Treatment of membranes with 5'-guanylylimidodiphosphate prior to incubation with Au(III) prevented the inhibitory effect of Au(III) on adenylate cyclase. Our data suggest that Au(III) alters the response of adenylate cyclase to agonists most likely through interaction with specific sulfhydryl groups associated with the enzyme system.     

Change in the adenylate cyclase activity of the lymphocytes and thrombocytes in irradiated sheep

Changes in basal and prostaglandin stimulated activity of adenylate cyclase in lymphocytes and platelets of irradiated sheep are phasic. In platelets adenylate cyclase activity is maximum on days 5 and 10 following irradiation, whereas in lymphocytes the enzyme activity is maximum on day 7. cAMP phosphodiesterase activity is slightly changed in platelets and increased in lymphocytes of exposed sheep.     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Regulation of platelet adenylate cyclase by adenosine.

The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5'-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Desensitization of prostaglandin-activated platelet adenylate cyclase

Prostaglandin D₂ (PGD₂) is one of several prostaglandins that can inhibit platelet aggregation and activate adenylate cyclase. Platelets were exposed to varying concentrations of PGD₂, washed, and the adenylate cyclase response to prostaglandins, epinephrine, and sodium fluoride determined. Incubating platelets with 5 × 10^?5 M PGD₂ for 2 hr resulted in a 45% decrease in PGD₂ activation of adenylate cyclase and a 25% decrease in stimulation by PGE₁. Fluoride activation (7-fold) epinephrine inhibition (30%) and basal enzyme activity were unchanged by exposure of the platelets to PGD₂. Desensitization was concentration dependent, with loss of enzyme activity first noted when platelets were incubated with 10^?7 M PGD₂. Enzyme sensitivity could be partially restored when desensitized platelets were washed free of PGD₂ and incubated in buffer for 2 hr; complete resensitization required incubation for 24 hr in plasma. Regulation of prostaglandin sensitive platelet adenylate cyclase could be of importance in mediating the response of platelets to aggregating agents.     

Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.     

The epinephrine-sensitive adenylate cyclase of rat liver plasma membranes. Role of guanyl nucleotides.

The epinephrine sensitivity in vitro of the adenylate cyclase system in liver plasma membranes from adrenalectomized rats was increased by the addition of 1 to 100 muM GTP or GDP in the incubation medium. Basal and glucagon-stimulated cyclase activities were also enhanced by GTP and GDP. These effects occurred even in the absence of an ATP-regenerating system. They were mimicked by 5'-guanyl diphosphonate and a series of guanyl derivatives, indicating that the structural requirement for the GTP action is not very stringent. Guanyl nucleotides did not increase the affinity of the adenylate cyclase system for the activating hormones, nor did they protect the enzyme activity against denaturation. Their synergic effect was due to an enhancement of the affinity of the enzyme for the substrate MgATP and also to an increase of the maximal velocity of the reaction. It is proposed that the guanyl nucleotides act directly and primarily upon the catalytic component of the cyclase system, independently of their effects on the binding of the activating hormones to liver plasma membrane. Since the activating effects of epinephrine and glucagon are similar in the presence of GTP, but not in its absence, it is suggested that the lower efficiency of epinephrine under normal conditions is not due to intrinsic membrane characteristics, but rather, to superimposed extraneous modulations.