| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide that we called P47 of <M_r> 47,000 (Imaoka, T. and Haslam, R.J., J. Biol. Chem. 258, 11404, 1983), by protein kinase C. Since the identity and function remains to be known, we purified protein kinase C, unphosphorylated and phosphorylated P47 to homogeneity from human platelets. Then precise phosphorylation reaction of P47 in vitro and a biological function of P47 were studied. Protein kinase C catalysed the phosphorylation reaction of P47 protein, platelet myosin light chain, histone <III> -S with Km of 0.8 <-!+> 0.2, 4.2 <-!+> 0.5, 4.7 <-!+> 0.7 <micro> M, and Vmax of 0.312, 0.189, 0.874 <micro> mole/min/mg, respectively. Some data previously obtained in this laboratory and others utilizing histone <III> -S as substrate are consistent with the synergistic effect by diacylglycerol (DG) in the presence of <Ca^(++)> , phosphatidylserine (PS). Using P47 as substrate, the enz
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yme required both <Ca^(++)> and PS, but not DG for activity.
<^(125)I> labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presense of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had a inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8(P47:actin). These activities were <Ca^(++)> independent. Purified <^(32)P> -labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization.
Therefore, we propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction. On the other hand, whether DG in fact can act as a second messenger remain uncertain. (supported by MESC of Japan) Less
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