Characterization of a calmodulin-dependent protein phosphatase from human platelets.
A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.
Tallant
E A
EA
Wallace
R W
RW
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D001921:Brain; D002135:Calcium-Binding Proteins / Q000097:blood*; D002147:Calmodulin / Q000302:isolation & purification; D002148:Calmodulin-Binding Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000097:blood* / Q000302:isolation & purification; D013552:Swine; D014268:Trifluoperazine / Q000494:pharmacology; D014336:Troponin / Q000494:pharmacology; D019209:Troponin C; D014357:Trypsin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007093:Imidazoles; D046911:Macromolecular Substances; D014336:Troponin; D019209:Troponin C; D014268:Trifluoperazine; C031938:calmidazolium; D010749:Phosphoprotein Phosphatases; D014357:Trypsin