Catalytic Activity Of Adenylate Cyclase Research Articles

The prostaglandin antagonist polyphloretin phosphate behaves as a nonspecific inhibitor of rat hepatic adenylate cyclase

The mechanism of the nonspecific inhibitory effects of the prostaglandin antagonist polyphloretin phosphate (PPP) was studied on a partially purified adenylate cyclase preparation from rat livers, PPP in concentrations above 100 μg/ml reduced the enzyme activity below basal values regardless of the stimulator used. The activity of adenylate cyclase in a persistent active state due to enzyme preincubation with guanylylimidodiphosphate (Gpp(NH)p) was inhibited 5–10 times more weakly. Kinetic studies indicate that PPP does not influence directly the catalytic site for ATP. PPP seems to produce permanent enzyme inhibition which is not antagonized by repeated washing of the enzyme preparation. The reduced catalytic activity of adenylate cyclase retains after treatment with PPP the character of tis responsiveness to the stimulators, which depends on the state of the enzyme before exposure to the inhibitor.

Coupling of catecholamine receptor from one cell with adenylate cyclase from another cell by cell fusion.

The experiments test the hypothesis that beta-adrenergic receptor is an independent unit that can be transferred from one adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4-6-1-1[ system to another. Turkey erythrocytes in which the catalytic activity of adenylate cyclase had been inactivated by N-ethylmaleimide or by heat contributed the beta-adrenergic receptor. Friend erythroleukemia cells (F cells) that possessed no measurable beta-adrenergic receptor contributed the adenylate cyclase. The erythrocytes in which the enzyme had been inactivated were fused with the F cells by Sendai virus. The cell ghosts of the fused preparation demonstrated adenylate cyclase activity which was strikingly enhanced by isoproterenol. Controls of fusion of F cells with each other or with human erythrocytes failed to show a response to isoproterenol. It was therefore concluded that the beta-adrenergic receptor of the turkey erythrocytes must have become functionally coupled to the adenylate cyclase of the mouse F cells. Activation by isoproterenol was demonstrable within a few minutes after fusion, and inhibitors of protein synthesis had no effect. Thus, coupling must have occurred between the preexisting components. The findings suggest that it may be possible in the future to confer on cells that possess an adenylate cyclase system new hormonal responses by inserting a receptor into their cell membrane. It is proposed that the procedure of massive heterologous cell fusion, as used in the present study, can be used to analyze the function of other cell membrane components.

Characterization of 125I-Glucagon Binding in a Solubilized Preparation of Cat Myocardial Adenylate Cyclase: FURTHER EVIDENCE FOR A DISSOCIABLE RECEPTOR SITE

Abstract A solubilized preparation of cat myocardium, which contains adenylate cyclase, has been shown to specifically bind biologically active 125I-glucagon. 125I-glucagon binding was observed over the concentration range 1 x 10-7 to 1 x 10-5 m. Unlabeled glucagon displaced 125I-glucagon over a similar concentration range. The binding specificity of this preparation was also shown by the fact that parathyroid hormone did not bind to cardiac receptor sites in this preparation nor did it displace 125I-glucagon from its binding site. The binding of 125I-glucagon at 37 or 25° is linear for approximately 30 min until maximum binding is reached. In contrast, the activation of adenylate cyclase is maximal within 5 min, indicating the presence of additional glucagon binding sites over and above those required for activation of the enzyme. The binding material was stable at 4° for 4 days and indefinitely when stored in liquid nitrogen. Boiling the binding material for 15 min or incubating it with 1 n HCl for 15 min destroyed most of its glucagon-binding ability. Optimal binding was observed over a broad pH range from 3.6 to 8.5, with a decline above pH 9.0. Preincubation of the binding material with trypsin decreased binding about twothirds. Phospholipases A and C, DNase, RNase, neuraminidase, urea (1 m), GTP, and ATP were without effect on the binding. Solubilized myocardial adenylate cyclase has been shown to have a molecular weight of about 100,000 to 200,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the crude solubilized preparation incubated with 125I-glucagon suggested a molecular weight for the binding fraction of approximately 26,000. Chromatography of the 125I-glucagon-receptor complex on either Sephadex G-100 or Bio-Gel P-30 produced a dissociation of the receptor from catalytic adenylate cyclase activity, which apparently represents a larger molecular weight component (g 100,000). The 125I-glucagon-receptor complex eluted from the gels at an elution volume consistent with the salt peak. However, this eluate was shown by hydrodynamic flow electrophoresis to be neither free glucagon or iodine. Moreover, when it was subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gels, the complex had a molecular weight of about 26,000, identical with what was obtained with the crude material. It appeared, therefore, that the complex of glucagon to its receptor had a strong positive charge which resulted in adsorption of the complex to the gel. Following neutralization of the charge-gel interaction by Sephadex and Bio-Gel chromatography of the 125I-glucagon-receptor complex in 0.01 or 0.025 n NaOH, the binding fraction eluted in a more appropriate location for its apparent size. The property of adsorption to Sephadex G-100 and Bio-Gel P-30 may prove useful in purification of the glucagon receptor site.

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.

Expression of adenylate cyclase, catecholamine receptor, and cyclic adenosine monophosphate-dependent protein kinase in synchronized culture of Chang's liver cells (S phase-membrane receptors-NaF stimulation).

In Chang's liver cells synchronized by exposure to thymidine, both cyclic adenosine monophosphate-dependent and cyclic adenosine monophosphate-dependent and cyclic adenosine monophosphate-independent protein kinase activities increased throughout the cell cycle in a manner that corresponded approximately to the overall increase in cellular protein. Thus, the catalytic and the cyclic adenosine monophosphate-binding regulatory subunits of protein kinase appear to be temporally coexpressed. Adenylate cyclase activity stimulated by catecholamine, as well as basal activity, decreased markedly after release of cells from thymidine blockade during the S phase of the cell cycle; this was followed by recovery and overall net increase to fully expressed activity by the peak of mitosis. After initial decrease early in the S phase, cyclase activity assayed in the presence of fluoride ion began to rise before the rise in basal and catecholamine-stimulated activities, indicating that regulation of membrane receptors for catecholamines and for basal activity may be at least in part separate from that for catalytic activity of adenylate cyclase. The differential expression of membrane receptors for adenylate cyclase during the Chang's liver cell cycle affords a possible mechanism for control of response to hormones during the cell cycle.