Human complex-forming glycoprotein, heterogeneous in charge: the primary structure around the cysteine residues and characterization of a disulfide bridge

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of $\text{1}\text{v}$ versus $\text{1}\text{ATP}$. Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low K_m (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg²⁺, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K_m enzyme.