Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5'-(gamma-thio) triphosphate.

By SDS-polyacrylamide gel electrophoresis, mitochondrial proteins having covalently-bound flavin were analyzed. Mitochondria were prepared from the liver of rat injected with radioactive riboflavin. Radioactivity was found to be associated with four protein components. Their subunit molecular weights were 91,000, 72,000, 60,000 and 44,000. The first two components exhibited yellowish fluorescence on a gel under ultraviolet illumination. The component of the highest molecular weight seems to be a new protein containing covalently-bound flavin.     

Specific MG2+ and adenosine sites involved in a bireactant mechanism for adenylate cyclase inhibition and their probable localization on this enzyme's catalytic component.

The specificity of adenosine sites involved in adenylate cyclase inhibition (P sites) is identical on membrane-bound and on solubilized enzyme. Kinetic analysis indicates that in addition to a low affinity Mg²⁺ site involved in adenylate cyclase stimulation (Km = 10 mM), there is a high affinity Mg²⁺ site (Km = 2.10^?4M) involved together with P sites in a bireactant mechanism for triggering adenylate cyclase inhibition. Guanyl nucleotide-binding protein does not seem to be implicated in this inhibition. We were not able to separate the catalytic component of adenylate cyclase from P sites, either on a sucrose density gradient or in gel filtration experiments. It is suggested that P sites are located on the catalytic component of the enzyme.     

The role of GTP in the activation of adenylate cyclase.

An attempt is made to integrate the knowledge on the role of hormones and guanyl nucleotides in regulating adenylate cyclase into a single molecular model. It is suggested that the hormone catalyzes the activation of the enzyme adenylate cyclase by facilitating the conversion of the enzyme from its inactive state to its active form. The hormone is also responsible for the termination of the signal namely the deactivation of the enzyme by inducing the hydrolysis of GTP at its regulatory site. The relative rates of these two processes determine the steady state concentration of the active form of the enzyme. The model also explains the difference in behaviour between GTP and its non-hydrolyzable analogs GppNHp and GTPγS.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.     

Cyclic AMP metabolism in mouse parotid glands properties of adenylate cyclase,protein kinase and phosphodiesterase

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands.Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity with a pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a K_a of 1.2·10^?7 M. Parotid cyclic AMP and cyclic GMP phosphoriesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least six peaks of enzyme activity in the pI range of 4–6.Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine ′,5′-monophosphatase (cyclic AMP) · 2 h^?1 · mg^?1 protein. This enzymatic activity is strongly stimulated by NaF and 5′-guanylimidodiphosphate, is slightly stimulated by epinephrine, norephrine, soproterenol, and prostaglandin E, and is inhibited by calcium. The hormone stimulation is not potentiated by 5′-guanylylimidodiphosphate.     

Isolation of homologous and heterologous complexes between catalytic and regulatory components of adenylate cyclase by forskolin-Sepharose

Homologous and heterologous complexes between catalytic and GTP-binding components can be isolated by means of immobilized succinyldeacetylforskolin (forskolin-Sepharose). A heterologous complex is formed by reconstitution of forskolin-Sepharose bound catalytic function from rabbit myocardial membranes with the homogenous [3H]methyl-GTP-binding protein from duck erythrocyte membranes. Analysis of the reconstituted complex by sodium dodecyl sulfate polyacrylamide gelelectrophoresis reveals that only the Mr 42 000 component of the GTP-binding protein's Mr 42 000/Mr 35 000 heterodimer contributes to the formation of active adenylate cyclase.     

Calmodulin activates adenylate cyclase from rabbit heart plasma membranes

It was shown that calmodulin (CM) activates the adenylate cyclase (AC) of rabbit heart light sarcolemma in the presence of micromolar free Ca2+ concentrations and this effect is blocked by trifluoroperazine and troponin I. GTP (in the presence of isoproterenol) and Gpp(NH)p are able to increase the CM-dependent activity of enzyme. It was concluded that there is no special CM-dependent "form' of AC in the heart and the common catalytic component of AC can be regulated both by CM and guanine nucleotide-binding regulatory component (N-protein). In the presence of Ca2+ and guanine nucleotide heart AC exists as a complex: CM-catalytic component-N-protein.     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans I. Inhibition by heparin of gonadotropin- stimulated ovarian adenylate cyclase

Heparin inhibits (I₅₀ = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Guanine nucleotides protect adenylate cyclase against inhibition by Pb2+

The inhibition of rat liver adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Pb²⁺ could be separated into an irreversible and a reversible component.Evidence was obtained that both types of inhibition were due to free Pb²⁺, rather than Pb/ATP, and that Pb²⁺ did not act via the site wherein Mg²⁺ and Mn²⁺ activate the cyclase.Guanine nucleotides strongly counteracted the reversible inhibition of cyclase by Pb²⁺, providing onother example of guanine nucleotide effects on adenylate cyclase function.It is suggested that the Pb²⁺-inhibited cyclase may be of value in the study of guanine nucleotide-cyclase interactions.     

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the K_a was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K_D 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β₂ antagonist butoxamine; the β₁ antagonist practolol was inactive. [³H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β₂ type.     

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.