Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

1981 ◽  
Vol 91 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R M Lewis ◽  
D L Nelson
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Excitable Membrane ◽  
Protein Kinase Activity ◽  
Paramecium Tetraurelia ◽  
Phosphoprotein Phosphatase ◽  
Endogenous Protein ◽  
Endogenous Substrates

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.

Activation of protein kinase activity in pancreatic acini by calcium and cAMP

1984 ◽  
Vol 246 (5) ◽  
pp. G500-G508 ◽  
Author(s):  
D. B. Burnham ◽  
J. A. Williams
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Free Calcium ◽  
Protein Kinase Activity ◽  
Inhibitor Protein ◽  
Pancreatic Acini ◽  
Protein Substrates ◽  
Phosphorylated Proteins ◽  
Maximal Activation ◽  
Activity Dependent

Regulation of protein kinase activity by calcium and cAMP was investigated in cytosolic and particulate preparations from isolated mouse pancreatic acini. In cytosol, three protein kinase activities could be distinguished: a calcium-activated kinase activity that was increased by exogenous calmodulin (CaM) and abolished by treatment of cytosol with a phenothiazine-coupled resin, a calcium-activated kinase activity dependent on phosphatidylserine (PS), and cAMP-activated kinase activity. Phosphorylation of a Mr = 92,000 cytosolic protein was greatly increased by both CaM-dependent and cAMP-activated kinases, whereas PS-dependent kinase activity most heavily phosphorylated proteins of Mr = 62,000 and 40,000. In addition, these kinase activities demonstrated differences in specificity for exogenous protein substrates. CaM-and PS-dependent kinases were completely blocked by trifluoperazine; the inhibitor protein of cAMP-activated protein kinase selectively inhibited cAMP-activated kinase activity. Exogenous CaM decreased the concentration of free calcium for half-maximal activation of CaM-dependent kinase activity from 5.5 +/- 0.5 to 1.2 +/- 0.3 microM Ca2+; half-maximal activation of the PS-dependent and cAMP-activated kinase activities was achieved at 12 microM Ca2+ and 40-50 nM cAMP, respectively. In a particulate fraction depleted of endogenous CaM, CaM-dependent and cAMP-activated kinase activities were detected. In conclusion, this study demonstrates the existence of protein kinases in pancreatic acini which may be involved in the action of pancreatic regulatory agents that use calcium or cAMP as an intracellular messenger.

Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase

1976 ◽  
Vol 54 (5) ◽  
pp. 438-445 ◽  
Author(s):  
Prakash V. Sulakhe ◽  
Nicholas Ling-Kit Leung ◽  
Patrick J. St. Louis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Cardiac Sarcolemma ◽  
Guinea Pig Heart ◽  
Calcium Accumulation ◽  
Exogenous Protein ◽  
Endogenous Protein ◽  
Membrane Phosphorylation

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmai calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.

scholarly journals Protein kinase activity associated with pancreatic zymogen granules

1985 ◽  
Vol 227 (3) ◽  
pp. 743-751 ◽  
Author(s):  
D B Burnham ◽  
P Munowitz ◽  
N Thorn ◽  
J A Williams
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
High Speed ◽  
Kinase Activity ◽  
Integral Membrane Proteins ◽  
Protein Kinase Activity ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Protein Substrates ◽  
Amp Activated Protein Kinase

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.

scholarly journals A phospholipid-stimulated protein kinase from Dictyostelium discoideum

1989 ◽  
Vol 260 (2) ◽  
pp. 557-561 ◽  
Author(s):  
B Jiménez ◽  
A Pestaña ◽  
M Fernandez-Renart
Keyword(s):  
Protein Kinase ◽  
Protease Inhibitors ◽  
Dictyostelium Discoideum ◽  
Kinase Activity ◽  
Deae Cellulose ◽  
Protein Kinase Activity ◽  
Endogenous Protein ◽  
Exogenous Substrate ◽  
Intermolecular Reactions

A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme.

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