Phosphorylation of endogenous proteins by endogenous protein kinase of density gradient--purified plasma membrane vesicles of Ehrlich cells.

A phosphorylation of endogenous acceptor protein(s) has been demonstrated to occur in membraneous vesicles prepared from Ehrlich ascites tumour cells. The reaction was catalyzed by endogenous protein kinase in the presence of exogenous (gamma32P)ATP. A considerable increase of the specific protein kinase activity took place when the plasma membrane preparation was subjected to a further gradient centrifugation in Dextran T 150. This was done in the presence of a slightly alkaline phosphate buffer containing Mg-ions which resulted in the formation of a well defined vesicular preparation at density 1.035 in the gradient. The apparent Km and Vmax for the reaction with vesicles and exogenous (gamma32P)ATP were determined and found to be 0.022 mM and 0.23 nmol x mg-1 x 10 min-1, respectively. Neither cyclic AMP nor cyclic GMP did stimulate the protein kinase-catalyzed reaction. Instead, a clear inhibition of the reaction by the cyclic nucleotides was unexpectedly registered. Adenosine at 0.5 mM also inhibited the reaction. Calcium ions were inhibitory at all concentrations tested in the presence of a fixed (gamma32P)ATP/Mg2+ ratio. When Mg-ions were stoichiometrically replaced by Ca-ions practically no activity was observed.