Journal of cyclic nucleotide and protein phosphorylation research最新文献

The activities of two cyclic AMP phosphodiesterases of baker's yeast (Saccharomyces cerevisiae) were measured during diauxic batch growth on 2% glucose. The specific activity (units/mg of yeast protein) of the Mg-independent, high Km phosphodiesterase increased 20-fold throughout the 108 h cultivation. The specific activity of the Mg-dependent, low Km phosphodiesterase about doubled during glucose utilization and fell back to the initial level as the cells entered stationary phase.

Since there is evidence suggesting that nicorandil (SG-75) relaxes coronary arterial smooth muscle by increasing cGMP levels, the effects of this vasodilator on soluble guanylate cyclase from bovine coronary arteries were studied more closely. It was found that nicorandil stimulated guanylate cyclase dose-dependently (3-30 mM) up to 100-fold the control value. Similar to nitroglycerin but in contrast to sodium nitroprusside, cysteine (0.5-20 mM) was required to obtain this stimulation. All other investigated thiols, except thiosalicylic acid which was partially able to mimic the cysteine effect, were ineffective. As evident from time course studies, nicorandil induced stimulation of guanylate cyclase was characterized by a lag-phase which could be avoided by preincubating the enzyme with nicorandil. The stimulatory effect of nicorandil was diminished in the presence of methylene blue, ferricyanide or hydroquinone. These results give further evidence that a) nicorandil exerts its vasodilating effect via stimulation of guanylate cyclase and b) nitrate esters, such as nitroglycerin or nicorandil, stimulate the enzyme, at least in vitro, only in the presence of cysteine or, to a lesser extent, thiosalicylic acid.

Alloxan exerts a selective impairment of the insulin-producing B-cells of the islets of Langerhans, which may result in diabetes mellitus. The effects of alloxan on cyclic AMP metabolism in isolated mouse islets of Langerhans were investigated. Alloxan caused an immediate increase in islet content of cyclic AMP, whereas a subsequent glucose-stimulated increase of islet cyclic AMP content was inhibited in alloxan-exposed islets. No corresponding effects of the drug were, however, found on either islet adenylate cyclase or cyclic AMP phosphodiesterase activities in broken cell preparations. It appears unlikely that there is a direct interaction between alloxan and the enzyme molecules leading to irreversible changes. Alloxan may rather affect some metabolic factor essential for optimal enzyme function. The inhibition of glucose-stimulated increase in islet ATP content and adenylate energy charge in alloxan-treated islets suggests that such a factor might be dependent on intact ATP generation.

beta-Adrenergic receptors were determined in sixteen cultured tumor cell lines of T- and B-lymphoid derivation by the binding of the hydrophilic ligand [3H]CGP-12177. The number of receptors per cell varied from less than a hundred to over three thousand. Intracellular cAMP responses to isoproterenol and forskolin were measured for seven cell lines possessing a range of receptor numbers. No direct relationship was found between the isoproterenol-induced elevation of cAMP and the receptor number, and forskolin-induced increases in cAMP varied by more than 100-fold between cell lines. Adenylate cyclase activity was also measured in membrane preparations from the seven cell lines. The T-lymphoma line WEHI-22, the line with the highest receptor number (3700 per cell), showed very little response to any stimulatory agent. With the other four cell lines possessing receptors, the membrane-bound adenylate cyclase activity evoked by isoproterenol relative to that evoked by forskolin was found to be proportional to the number of receptors per cell. The number of receptors thus seems to determine the proportion of the inherent adenylate cyclase that can be activated by a beta-adrenergic agent. The levels of adenylate cyclase induced in membranes by GppNHp and by fluoride, relative to those induced by forskolin, were substantially higher with some cell lines than with others. These data and the results from WEHI-22 cells indicate that lymphoid cell lines differ in their content of functional C and N proteins.

The activation of adenylate cyclase by forskolin was investigated in terms of the dissociation model of guanyl nucleotide binding protein (Ns). It was demonstrated that the biphasic forskolin concentration-response of adenylate cyclase could be explained by the dissociation of the beta subunit. The equations developed from such a theoretical approach gave an accurate description of concentration response data from S49 cultured mouse lymphoma cells.

We have used the indirect colloidal immunogold technique to examine the ultrastructural localization and distribution of the regulatory subunits (RI and RII) and catalytic subunit (C) of cAMP-dependent protein kinase (cA-PK) in hepatocyte nuclei during the prereplicative phase of rat liver regeneration. The technique allowed the localization of all three subunits of cA-PK in hepatocyte nuclei. Morphometric quantitation of the relative staining intensity of nuclear antigen indicated a marked increase of immunogold staining of nuclear RI and C 16 hours following partial hepatectomy. In contrast, staining of nuclear RII was not affected. These immunocytochemical observations correlate well with our previous data (Laks, M.S. et al. (1981) J. Biol. Chem. 256, 8775-8785) obtained by biochemical methods indicating a modulation of cA-PK activity after partial hepatectomy.

Forskolin, a diterpene, has been reported to reversibly stimulate adenylate cyclase from a number of mammalian tissues. Adenylate cyclase preparations derived from sperm are reported to be both deficient in the guanine nucleotide subunit and insensitive to forskolin. Despite the latter observation, we report here that forskolin elevates cAMP levels in immature caput sperm, and initiates motility in the presence of "permissive" agents such as bicarbonate, procaine and dibucaine. In mature caudal sperm forskolin stimulates motility in a concentration dependent manner in the presence of the phosphodiesterase inhibitor IBMX but elevates cAMP levels only briefly before nucleotide levels return to control values. In addition, forskolin stimulates adenylate cyclase activity associated with plasma membrane preparations of caput sperm but not caudal sperm. This differential action of forskolin on these cell types could provide a basis for understanding the regulation of adenylate cyclase in both immature and mature bovine sperm.

Methods specially designed for the assay of cyclic adenosine 3':5'-monophosphate (cAMP) in human saliva have not previously been published. Methods for measurements in plasma or tissue preparations are inapplicable to saliva due to interference. This interference can be reduced by calibrating standard solutions with reagent blanks prepared from saliva pre-treated to remove cAMP (by charcoal adsorption). Further, human saliva contains only small amounts of cAMP (approximately 0.2 pmol/50 microliters saliva). The method presented provides the necessary sensitivity (lower detection limit: 0.02 pmol/50 microliters) obtained by adjustments of tracer and binding protein concentrations. The method is simple and has a comparatively great capacity for the number of test tubes to be assayed.

The ADP-hydrolyzing enzyme apyrase inhibited platelet aggregation and phosphorylation of a 40,000 dalton platelet protein (P40) induced by 1-oleoyl-2-acetyl glycerol (OAG), indicating a dependence on secreted ADP. Apyrase also enhanced OAG-induced potentiation of forskolin or prostaglandin I2 activation of cyclic AMP formation in platelets. Cyclic AMP formation induced by OAG alone could be demonstrated in the presence of a phosphodiesterase inhibitor. Elevation of cyclic AMP level inhibits platelet aggregation so that secreted ADP may be required to inhibit OAG-activated adenylate cyclase for aggregation to proceed. In contrast, apyrase only partially affected phosphorylation of P40 and aggregation induced by the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA caused marked inhibition of forskolin-stimulated cyclic AMP formation. TPA inhibition of cyclic AMP formation was largely reversed by apyrase, indicating that it was mainly due to release of ADP.