PROPERTIES OF PHOSPHATIDYLINOSITOL KINASE IN HUMAN PLATELETS

We have reported the specific ³²P-labelling in phosphatidyl-inositol-4-monophosphate(PIP) of intact platelets upon addition of the agents which elevate intracellular cAMP (Thrombos.Res.44, 155,1986).This event may be catalyzed by the action of Pl-kinase, the properties of which has not been elucidatedyet.Thereby, attempts were made to assay and to characterize PI-kinase of human platelets.Fresh lysed platelets prelabelled with ³²P in cold Tris-HCl buffer containing 2mM EGTA were incubated at 37 C in the presence of MgCl₂ for designated times and the phospholipids were extracted and analyzed by thin layer chromatography.³²P-labelling in PIP was gradually increased in consort with the decreased labelling in PI-4,5-bisphosphate.As the changes in the labelling was not affected by the presence of apyrase and as the radioactive inositol trisphosphate was not detected,it was suggested that the changes is due to the action of phoshomono-esterase rather than PI-kinase or phospholipase C.When ³²P-ATP was added to non-labelled lysed platelets upon incubation, ³²P was labelled only into PIP and the amount was markedly increased until 5min. after incubation.Since the labelling was strongly inhibited by apyrase,it likely reflects the activity of Pl-kinase. The activity of PI-kinase thus measured required Mg²⁺ strictly for the activity and the maximal activity was obtained in the presence of 30mM Mg²⁺ .In contrast,it was markedly inhibited in the presence of Ca²⁺ (as low as 2mM Ca²⁺ in the presence of 2mM EGTA),which was compatible.with our previous findings with intact platelets. The activity of A-kinase was not inhibited by a low concentration of Ca²⁺ .Furthermore,the activity was inhibited by cAMP or dbcAMP in a dose related manner and no enhancement of the activity Was obtained by the addition of catalytic subunit of A-kinase,though a significant reduction in the activity was observed in the presence of inhibitor protein to A-kinase. From these observations,the following conclusions were obtained; l)The activity of Pl-kinase in lysed platelets may be determined by pulse labelling with ³²P-ATP. 2)It requires Mg²⁺ absolutely and is inhibited by a very low concentration of Ca²⁺. 3)Pl-kinase is activated by A-kinase but the activated enzyme is inhibited by cAMP, suggesting the presence of feedback mechanism.