AFFINITY LABELING OF BOVINE BRAIN PROTEIN-KINASE-C BY TOSYL LYSYL CHLOROMETHANE - A KINETIC-STUDY

The kinetics of inactivation of bovine brain protein kinase C (PKC) by N(alpha)-p-tosyl L-lysyl Chloromethane (TosLysCH2Cl) were investigated. In absence of activators PKC gave non-linear semilog inactivation plots. At each reagent concentration a plateau of residual activity was reached after some time; its value was inversely proportional to TosLysCHCl concentration but the plateau was not due to inactivator depletion. On the other hand, in the presence of Ce2+, phosphatidylserine and phorbol 12-myristate 13-acetate, the activity loss followed saturation kinetics, with k(inact) = 0.6 x 10(-3) s-1 and K(inact) = 1.9 mM. The study of protection effects by ATP Mg2+ and histone required the presence of 50% glycerol in the incubation mixtures, otherwise the controls (kinase in the presence of activators and ATP Mg2+ or histone) rapidly lost activity . In the presence of 50% glycerol, the inactivation parameters were somewhat altered (k(inact) = 0.3 x 10(-3) s-1 and K(inact) = 0.2 mM); ATP Mg2+ proved to afford a mixed competitive-non competitive protection effect, while histone protected in a competitive manner with a K(p) of 0.06 mug/ml. In the presence and the absence of glycerol, plots of log k(obs) versus log inactivator concentration yielded straight lines with slopes of 0.7-0.9, indicating that 1 mol of reagent is sufficient for inactivation. The results described in this paper suggest that the reagent TosLysCH2Cl hits die catalytic domain of activated PKC at the active site, which is not available in resting PKC; in non-activated PKC, the labeling site would be different.