Activation of rat liver adenylate cyclase by guanosine 5′-[β,γ-imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein (original) (raw)
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Biochemistry, 1984
The stimulatory GTP-binding protein (G,) of adenylate cyclase, purified from rabbit liver, and 0-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of G, to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-0-(3-[3sS]thiotriphosphate) ( [3SS]GTPyS) binding to G, and ['2sI]iodocyanopindolol binding to receptors. Activation of reconstituted G, by GTPyS was stimulated up to 10-fold by the addition of the @-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [3sS]GTPyS could also be measured in the vesicles and was shown to parallel the functional activation of G, under all conditions. Most of the assayable G, in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTPyS binding were complete in less than 3 min, even under suboptimal conditions,
Activation of guanylate cyclase from rat liver and other tissues by sodium azide
Journal of Biological Chemistry, 1975
Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2-to 20.fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN, concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN, that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent K, for GTP from 35 to 113 pM. With NaN, activation the enzyme was less dependent upon the concentration of free Mn'+. Activation of enzyme by NaN, was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN, treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN, was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN,. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN,. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN,. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN,, whereas the soluble enzyme from these tissues was not. Little or no effect of NaNs was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN, on soluble guanylate cyclase from heart was probably due to the presence of an inhibitor of NaN, activation in heart preparations. The effect of NaN, was decreased or absent when soluble guanylate cyclase from liver was purified or stored at-20". The activation of guanylate cyclase by NaN, is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations. Recent reports suggest that guanosine 3':5'-monophosphate may act as a regulator of some biological processes (l-7). A number of agents may produce their effects in tissues by altering the accumulation of cyclic GMP' by either influencing the rate of synthesis by guanylate cyclase (EC 4.6.1.2) or the rate of hydrolysis by cyclic nucleotide phosphodiesterase (EC 3.1.4.17). The mechanism whereby some hormones and neurohormones increase cyclic GMP levels in tissues is unknown. From some analogies with the cyclic AMP system it has been assumed, rightly or wrongly, that such agents should alter guanylate cyclase activity. While some laboratories have
Histochemistry, 1985
Adenylate cyclase activity was demonstrated cytochemically in rat liver for the first time under the light microscope using cryostat sections mounted on glass cover slips and fixed with 1% glutaraldehyde for 1 min. Adenylate-(fl, y-methylene)diphosphate (AMP-P(CH2)P) was introduced as a new substrate for adenylate cyclase. It was found that adenylate cyclase was distributed heterogenously within the liver lobule. The enzyme activity was stronger in the area surrounding the central vein. A more specific localization at the plasma membrane and less unspecific background was obtained with AMP-P(CH2)P as compared to adenylylimidodiphosphate (AMP-P(NH)P). The specificity of the enzyme reaction using AMP-P(CH2)P was proved by increased formation of reaction product in the presence of 0.05 mg/ml glucagon and 0.125 mg/ml cholera toxin, as well as by inhibition of the reaction with 0.05 mg/ ml alloxan. These effects were also observed at the electron microscopic level. On the other hand, no increase in reaction was observed in the presence of glucagon with AMP-P(NH)P as a substrate for adenylate cyclase, and only a weak activation was observed after adding cholera toxin; alloxan-inhibition was not complete. These effects may be due to the presence of enzymes which hydrolyze AMP-P(NH)P nonspecifically, superimposing on the product of adenylate cyclase activity. We therefore suggest the use of AMP-P(CH2)P as substrate for histochemical adenylate cyclase demonstration in the liver.
Biochimica Et Biophysica Acta-biomembranes, 1976
1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P(NH)P yielded linear Arrhenius plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at .2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P.3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.
Brain Research, 1989
GTP-binding proteins (G proteins) have been implicated as mediators of several aspects of neuronal signal transduction including ion channels, phosphatidyl inositol turnover and the stimulation or inhibition of adenylate cyclase. Several investigators have employed the stable guanosine diphosphate (GDP) analog, guanosine 5"-O-thiodiphosphate (GDPflS) to block putative G protein-mediated processes. Although GDPflS is assumed to block G protein function, some investigators have reported partial activation of G protein-mediated processes by this compound. In this study we demonstrate that GDPflS functions as a partial agonist for the adenylate cyclase system. In rat cerebral cortex membranes, GDPflS activates adenylate cyclase with an ECs0 similar to the hydrolysis resistant GTP analog, guanylylimidodiphosphate (GppNHp), but to a far lower extent. Further, GDP/~S antagonizes the activation of adenylate cyclase by high doses of GppNHp or GTPyS (another stable GTP analog) but potentiates adenylate cyclase activation by low doses of these nucleotides. High doses of GDPflS provoke, only partially, exchange of nucleotides among G proteins, as measured by the transfer of the photoaffinity GTP analog, azidoanilido-GTP, between the inhibitory and stimulatory GTP-binding proteins. In the presence of the/~-adrenergic agonist, isoproterenoi, GDPflS fails to support stimulation of C6 glioma membrane adenylate cyclase and inhibits GppNHp-or GTPyS-mediated stimulation of that enzyme. Inhibition of C6 membrane adenylate cyclase by GTP analogs is also blocked by GDPflS. Finally, in C6 cells made permeable with saponin, where the /~-adrenergic receptor is 'tightly coupled' to the adenylate cyclase system, GDPflS, GppNHp or GTPyS stimulate adenylate cyclase at high concentrations, but only in the absence of isoproterenol. When isoproterenol is added to these cells, GppNHp or GTPyS enhance adenylate cyclase activation, yet GDPflS acts only to block that process. Thus, GDPflS appears to function as partial agonist for the stimulatory adenylate cyclase G protein but only when an activated neuroreceptor is not coupled to that protein.
Distribution of Adenylyl Cyclase and Guanylyl Cyclase in Rat Tissues
Journal of King Abdulaziz University-Science, 2003
The specific activities of adenylyl and guanylyl cyclases, the cAMP and cGMP phosphodiesterase of rat kidney, liver, heart and brain were examined. Of all tissues, kidney has the highest adenylyl cyclase specific activities by mean (± SD) of (145 ± 14 pmol/min/mg protein) followed by that of heart (113 ± 21 pmol/min/mg protein), brain (101 ± 8 pmol/min/mg protein) and liver (92 ± 9 pmol/min/mg protein). The specific activity of particulate guanylyl cyclase was the highest in liver (26 ± 1 nmol/min/mg protein), but the soluble form predominated in kidney (40 ± 2 nmol/min/mg protein). In contrast to the other tissues examined, brain showed relatively high cAMP and cGMP phosphodiesterase activities. The kinetic properties of the adenylyl cyclases and cAMP phosphodiesterases were also investigated. The optimum pH for both types of activity was found to be at 7.4. Subcellular fractionation of the kidney to locate adenylyl cyclase activity revealed that both the mitochondrial and the microsomal fractions had higher specific activities than that of the nuclear fraction. Studies on the tissue distribution of cyclic nucleotide PDE activity as well as adenylyl and guanylyl cyclase showed that it is widely distributed in intra and extracellular and they have an important role in signal and nucleotide transdaction.
The Journal of biological chemistry, 1986
The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neithe...