Journal of cyclic nucleotide and protein phosphorylation research最新文献

Several hormones, including catecholamines, histamine, and prostaglandin E1, regulate the function of human mononuclear leukocytes (MNL) by stimulating the accumulation of cAMP. Isoproterenol-stimulated cAMP accumulation in MNL isolated and washed at 4 degrees is five times greater than in cells prepared at ambient temperature. The current study was aimed at understanding this difference. cAMP accumulation in MNL prepared at ambient temperature could not be increased by chilling the cells for 4 hours. Warming MNL prepared at 4 degrees for 30 min, however, reduced later isoproterenol-, histamine-, and PGE1-stimulated cAMP accumulation by 65-85% without altering forskolin-stimulated cAMP accumulation and without altering cellular viability or ATP content. In broken cell preparations, there was no difference in either adenylate cyclase or phosphodiesterase activities, and no difference in the binding of isoproterenol to the beta-adrenergic receptors. The reduction in isoproterenol-stimulated cAMP accumulation in warmed intact cells was reversed when the MNL were incubated with autologous leukocyte-depleted blood or with plasma. These data suggest the presence of one or more factors in plasma that enhances hormone-stimulated adenylate cyclase activity in intact MNL.

The intact rat adipocyte was used to investigate the possibility of common intermediates in the insulin stimulation of cyclic AMP phosphodiesterase and the beta-adrenergic/adenosine regulation of adenylate cyclase. A five minute incubation of the isolated adipocytes with insulin produced a 50-100% increase in the phosphodiesterase activity found in the particulate fraction of homogenates. The insulin stimulation was not impaired by the presence of either agonist or antagonists of the inhibitory adenosine receptor which acts on adenylate cyclase. Phosphodiesterase activation by insulin was also observable above the level of stimulation produced by the beta-adrenergic agent isoproterenol and forskolin. The validity of the enzyme activity measurements was supported by measurements of the hormonal actions on cyclic AMP levels within the cells. Possible crossover between the adenylate cyclase and phosphodiesterase regulation systems at a post-receptor site was investigated using adipocytes exposed to bacterial toxins specific for the modification of guanine nucleotide binding proteins. Both cholera toxin, which irreversibly activates Gs and pertussis toxin which inactivates Gi caused some stimulation of the phosphodiesterase activity and suppressed activation by isoproterenol, but neither toxin prevented the insulin stimulation of cyclic AMP phosphodiesterase. These results suggest, while common components may participate in the beta-adrenergic stimulation of both adenylate cyclase and phosphodiesterase, the mechanism of insulin activation of the phosphodiesterase does not involve the components of adenylate cyclase regulation.

Adenylate cyclase activity in renal papillary membranes was stimulated by both vasopressin and the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA). The stimulations mediated by the two receptors were additive at all concentrations and interacted differently with other AC-stimulatory factors viz cholera toxin, pertussis toxin and fluoride ion. Treatment of papillary tubules with cholera toxin increased cyclase activity from 4.5 +/- 1.5 to 110 +/- 9.1 (SE, n = 5) pmol/min/mg protein. Maximally effective concentrations of vasopressin increased activity in control preparations to 10.3 +/- 2.8 (an increase of 5.8 +/- 1.3). In cholera toxin treated preparations, vasopressin increased activity to 138.9 +/- 14.5 (an increase of 28.9 +/- 5.4, n = 5; p less than .01). Pertussis toxin increased activity to 9.1 +/- 3.0. The response to vasopressin was enhanced such that the absolute maximum increase in activity was 12.6 +/- 3.9 (n = 5; p less than .01). Addition of the two toxins together produced a greater than additive stimulation to 145 +/- 36. Maximum increase in activity caused by vasopressin was further enhanced to 48 +/- 13 (n = 5; p less than .01). In contrast, cyclase stimulation by NECA was additive with stimulations by the two toxins, separately and in combination. The NECA stimulation however, was enhanced in the presence of fluoride ion while the vasopressin stimulation was additive at all concentrations. Papillary membranes contained two different cyclase-stimulatory coupling proteins with alpha-subunits of MW's 46,600 +/- 450 (SE, n = 6) and 41,500 +/- 480 (SE, n = 6) as identified on SDS-polyacrylamide gel electrophoresis following cholera toxin labeling. Taken together, these data suggest that two adenylate cyclase-stimulatory coupling mechanisms with different properties are operative in renal papillary membranes.

The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose-6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2-e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide.

[32P]ADP-ribosylation of membrane proteins catalyzed by either cholera toxin or pertussis toxin was markedly enhanced by NADP+. The effect was concentration dependent; with 20 microM [32P]NAD+ as a substrate maximal enhancement was obtained at a concentration of 0.5-1.0 mM NADP+ for rabbit and guinea-pig liver membranes and 0.1 mM NADP+ for human erythrocyte membranes. NADP+ appears to act by inhibiting the degradation of NAD+ by NAD+-glycohydrolase (NADase) present in membrane preparations, probably as an alternate substrate for the enzyme. Among inhibitors tested (NADP+, isonicotinic acid hydrazide, imidazole, nicotinamide, L-arginine methyl ester and HgCl2) to suppress the enzyme activity, NADP+ was the most effective and, at 10 mM, inhibited hepatic NADase activity by about 90%. The effect of NADP+ was much greater than that of other known effectors of ADP-ribosylation such as Mg2+ and phosphate, or the NADase inhibitors, isonicotinic acid hydrazide and isonicotinamide. In membranes which contain substantial activities of NADase the inclusion of NADP+ in the assay system is necessary to achieve maximal ADP-ribosylation of membrane proteins.

A procedure for measuring cyclic AMP in samples containing interfering substances, especially high concentrations of other adenine nucleotides, is described. Samples are purified by sequential chromatography on phenyl boronate followed by Dowex - 1. The final elution is with 0.1 normal hydrochloric acid; the samples are immediately ready for acetylation and radioimmunoassay. Cyclic AMP can be detected at 0.5 nanomolar without need to concentrate the sample at any step. The method has been successfully applied to samples containing 100 micromolar ATP, ADP, or AMP in tissue culture medium.

Digitonin-permeabilized monolayers of C6-2B rat astrocytoma cells exhibit adenylate cyclase activity in the presence of exogenously added ATP. The adenylate cyclase retains the qualitative and quantitative characteristics of hormone stimulated cyclic AMP accumulation in whole cells including GTP dependency and 100 fold stimulation by isoproterenol. Forskolin increased enzymatic activity in the absence of added GTP, however forskolin efficacy and potency was enhanced by GTP. Low non-efficacious concentrations of forskolin, without added GTP, supported isoproterenol-stimulated cyclase activity. The GTP-stimulated isoproterenol response was potentiated by forskolin. Forskolin support of isoproterenol stimulated cyclase in the absence of GTP raises the possibility that forskolin can act independently of GTP in coupling receptors to cyclase catalytic units and/or that forskolin could increase the efficacy and potency of GTP in the coupling reaction. Permeabilization of C6-2B and other cultured cells yields a preparation of adenylate cyclase which retains the enzyme in a state which closely approximates its activity in the native membrane--a system which could prove useful in studies of the regulation of adenylate cyclase in vivo.

The calmodulin sensitive phosphodiesterase of porcine cerebral cortex was characterized in terms of kinetic behavior, calmodulin activation, and stability. This enzyme displayed non-Michaelis-Menten kinetics in the presence or absence of calmodulin. The apparent affinity for cyclic GMP was higher than that for cyclic AMP but at saturating levels of substrate, this enzyme catalyzed the hydrolysis of cyclic AMP at a greater rate than it did cyclic GMP. The affinity of this enzyme for calmodulin was about 20-fold lower than usually reported. The apparent loss of phosphodiesterase activity after storage was found to be due to a strong association with container surfaces and could be prevented or reversed by the presence of 0.1% Triton X-100.