Two phosphorylated forms of myosin in thrombin-stimulated platelets.
Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.
Naka
M
M
Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Japan.
Saitoh
M
M
Hidaka
H
H
D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D009218:Myosins / Q000032:analysis*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D011493:Protein Kinase C; D013917:Thrombin; D009218:Myosins; D013755:Tetradecanoylphorbol Acetate