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.
