Identification, purification, and partial characterization of plasma protein kinases

Abstract

Two isomeric forms of protein kinases, FI and FII, were isolated from human plasma. These two isomeric enzymes were isolated to apparent homogeneity on NaDodSO 4-PAGE by using (NH 4) 2SO 4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue, and high-pressure liquid column chromatography. Polyclonal antibodies were obtained from immunized rabbits and both enzymes cross-reacted with each other. Furthermore, immunoaffinity-purified anti-FI and anti-FII antibodies inhibited the enzyme activity of both FI and FII. These enzymes are cyclic nucleotides, Ca 2+, calmodulin and phosphatidylserine-independent enzymes which can phosphorylate exogenously added histone, casein, protamine, phosvitin, and platelet surface proteins. The phosphorylated proteins of intact platelets by these enzymes in the presence of exogenously added [γ- 32P]ATP ranged in apparent molecular weights from 13.5K to 200K, as estimated by their mobility during NaDodSO 4-PAGE. Trypsin removed the label from the platelet surface phosphoproteins without affecting the intracellularly located phosphoproteins labeled endogenously by 32PO 4-prelabeling of intact platelets. These observations raise the possibility that these enzymes could play a role in modulating the properties of platelets through phosphorylation of the platelet surface proteins.