Journal of cyclic nucleotide and protein phosphorylation research最新文献

Prostaglandin A1 inhibits the energy-dependent export of cyclic AMP from avian red cells. When exposed to PGA1, avian red cells rapidly accumulate the prostaglandin and convert it to a polar metabolite. Inhibition of cyclic AMP efflux correlates with the intracellular content of this metabolite. Data in the literature and our chemical and chromatographic analyses indicate that the prostaglandin metabolite is a glutathione adduct of PGA1. Thus, we hypothesize that a GSH adduct of PGA1 acts intracellularly to elevate cyclic AMP by blocking its extrusion.

Previous studies demonstrated that opiate inhibition of adenylate cyclase (AC) in striatal membranes is related to an opiate-stimulated GTPase with a low Km. Dopamine (DA) also dose-dependently activates a high affinity GTPase, with a pattern of stimulation and a receptor selectivity (D1 type) similar to those observed in DA activation of striatal AC. Moreover, the DA- and the opiate-sensitive GTPase activities have different sensitivities to agents that affect the inhibition of AC, such as Na+ and N-ethylmaleimide (NEM), or the stimulation, such as cholera toxin (CTX). Thus, the impairment of opiate-dependent inhibition of AC in the absence of Na+ ions or after NEM pretreatment of the membranes is parallel with preferential impairment of the opiate-dependent GTPase. On the contrary, selective blocking by CTX of the DA-dependent GTPase leads to the enhancement of AC stimulation by DA. These results suggest that DA activation of striatal AC is related to a GTPase that is specifically stimulated by DA and is associated with the Ns protein. A distinct Ni protein seems to be responsible for the opiate effect on AC and GTPase.

Rod outer segments (ROS) from bovine retinae were found to have high levels of calcium/phospholipid dependent protein kinase (protein kinase C). Protein kinase C behaves as an extrinsic membrane protein and phosphorylates rhodopsin in a calcium-dependent manner. The abundance of protein kinase C in ROS is similar to that of rhodopsin kinase. Its ability to phosphorylate rhodopsin in ROS membranes suggests protein kinase C may play an important role in the regulation of signal transduction in the ROS. The limited set of extrinsic membrane proteins and abundance of protein kinase C makes this tissue an extremely useful source to purify protein kinase C. The extrinsic membrane protein fraction has 6-7 U protein kinase C activity per mg protein, and the enzyme is quite stable apparently due to the lack of proteases in the preparation. A procedure was developed using phosphatidylserine- and calcium-dependent binding of protein kinase C to phenyl-Sepharose in low ionic strength buffer to resolve protein kinase C and other calcium-binding proteins from the majority of extrinsic membrane proteins. Protein kinase C was eluted using EGTA, and peak fractions directly loaded onto a DEAE-cellulose column. The protein kinase C peak eluted from the ion-exchange column was pooled and had a specific activity greater than 1,000 nmol phosphate transferred to histone per min per mg protein with a recovery of 25 percent of the starting activity. The procedure to purify protein kinase C from ROS is simple and can be completed in one day.

Cholera toxin, injected into living fireflies, caused a delayed, non-hormone-dependent activation of adenylate cyclase, an elevation of cyclic AMP levels, and a brilliant persistent glow of the firefly light organ 8-20 hours following injection. During periods of spontaneous flashing, onset of toxin-induced glowing was abrupt and step-like in character, due to a markedly prolonged off-time of individual flashes. These observations provide a striking demonstration of the mechanism of cholera toxin action, and, together with other data, suggest that the initiation of the normal adult firefly flash is mediated through elevation of cyclic AMP levels secondary to the activation of an octopamine-sensitive adenylate cyclase.

The effects of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)- and (Rp)-cAMPS, on the kinetic properties of pyruvate kinase were studied in hepatocytes isolated from fed rats. Incubation of the cells with the cAMP-dependent protein kinase agonist, (Sp)-cAMPS, produced a concentration-dependent increase in S0.5 for phosphoenolpyruvate, but had no effect on Vmax. The (Sp)-cAMPS-treated enzyme was more sensitive to inhibition by alanine and ATP and, under the same conditions, was less responsive to activation by fructose-1,6-bisphosphate when assayed at a subsaturating phosphoenolpyruvate concentration. Incubation of the hepatocytes with only the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, produced no change in any kinetic parameters, but did suppress the (Sp)-cAMPS- or glucagon-induced increase in the S0.5 for phosphoenolpyruvate with IC50 values of 10 microM and 5 microM (Rp)-cAMPS. (Rp)-cAMPS is exerting an effect on the kinetic properties of pyruvate kinase through inhibition of cAMP-dependent protein kinase.

The regulation of adenylate cyclase by guanine nucleotides was examined in a plasma membrane preparation from Dictyostelium discoideum. At concentrations of greater than 10 microM, GDP, GTP and a non-hydrolyzable GTP analogue, guanosine 5'-(beta-gamma-imino)triphosphate (Gpp(NH)p), inhibited the enzyme. Guanosine, GMP and ITP were ineffective. The inhibition was not affected by variations in assay conditions (membrane concentration, time or temperature), the presence of cAMP, NaF or forskolin in the reaction mix, or variations in the stage of Dictyostelium discoideum development. There was no stimulation of adenylate cyclase by GTP or Gpp(NH)p under any conditions. Inhibition of adenylate cyclase by Gpp(NH)p was sensitive to divalent cations. The addition of MnCl2 resulted in increased adenylate cyclase activity, but augmented the inhibitory response to Gpp(NH)p. The differences between Dictyostelium discoideum and eukaryotic regulation of adenylate cyclase by guanine nucleotides are discussed.

The regulatory subunit (R) of Type I cAMP-dependent protein kinase from rabbit skeletal muscle can serve as a substrate for its catalytic subunit (C). The degree of phosphorylation depends on both the concentration of C and the the time of incubation. Moreover, the phosphorylation can be totally blocked by protein kinase inhibitors. In contrast, cAMP stimulates the phosphorylation of R using the holoenzyme. The purified holoenzyme isolated from rabbit skeletal muscle can be further fractionated into two fractions on DEAE Sephadex column. The first fraction eluted with low salt (50 mM NaCl) contains a much lower concentration of kinases than the second fraction eluted with high salt (100 mM NaCl), but the low salt kinase can be readily phosphorylated in the presence of MgATP. Our data thus implies that only a small fraction of Type I cAMP-dependent protein kinases in the skeletal muscle is present as the phosphorylatable species.

The effects of forskolin on phosphorylation of proteins of a 100,000 X g fraction was examined in isolated beating guinea pig hearts. Hearts were perfused with [32P] inorganic phosphate to label intracellular adenine nucleotides. Forskolin was injected into the coronary circulation and after freeze-clamping, phosphorylated proteins in a fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Forskolin increased the incorporation into a 25,000 Mr protein approximately 15 fold over control. Incorporation of label was time and dose dependent and was temporally coincident with increases in developed tension. A sarcolemmal fraction prepared from perfused hearts contained a similar 25,000 Mr protein. The data provides evidence that forskolin induced inotropy is accompanied by cAMP-dependent protein kinase mediated phosphorylation. The phosphorylation may be of the same protein whose phosphorylation is associated with epinephrine-induced increase in contractility.

In membranes of smooth muscle cells cultured from rabbit mesenteric artery, ethanol dose-dependently activates adenylate cyclase, both basal and PGE1- or GPP(NH)P-sensitive. The alcohol increases the maximal stimulation induced by PGE1 and GPP(NH)P, without greatly affecting their potency. The arrhenius plot displays a discontinuity point, which is only slightly lowered by ethanol. On the contrary, in membranes from human platelets ethanol inhibits basal, GPP(NH)P- and PGE1-sensitive adenylate cyclase, without modification of the prostaglandin or guanine nucleotide potency. The break point present in the Arrhenius plots is definitely lowered by the alcohol. In addition, ethanol decreases the thermostability of the enzyme. Neither in myocytes nor in platelets does ethanol affect the activation energy of the reaction. The data suggest that ethanol probably interacts directly with the membrane proteins, and that its effect is not mediated only through a perturbation of membrane lipid fluidity.

We have previously reported that the cAMP-specific phosphodiesterase activity in washed rat platelets is increased by a short exposure of platelet suspension to PGE1 and 1-methyl-3-isobutyl-xanthine (MIX). We report here that the incubation of washed platelets with forskolin resulted in an increase in the binding of cGMP and the activity of cGMP-phosphodiesterase as well as that of cAMP-specific phosphodiesterase. As for PGE1, MIX potentiated the stimulatory effect of forskolin. The maximal activation of phosphodiesterases by forskolin and MIX occurred after 30 sec of incubation of platelets (with a slow decline thereafter). The activation of phosphodiesterases in intact platelets by forskolin occurred in parallel with the dissociation of a cAMP-dependent protein kinase. Prior incubation of a platelet supernatant with Mg-ATP and cAMP had only a slight effect on cAMP- or cGMP-phosphodiesterase activities, but the presence of MIX during the prior incubation, followed by appropriate dilution, greatly enhanced the activity of the two phosphodiesterases. The phosphodiesterase activation in vitro was inhibited by a non-hydrolysable analogue of ATP, AMP-PNP. Since the cGMP-binding phosphodiesterase activity is enhanced by the catalytic subunit of cAMP-dependent protein kinase in the presence of MIX and absence of cAMP, the effect of MIX cannot be explained in terms of the protection of cAMP from hydrolysis. It is possible that the xanthine increases the susceptibility of the cAMP-specific and cGMP-binding phosphodiesterases to phosphorylation.