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 investigate d. In absence of activators PKC gave non-linear semilog inactivation p lots. At each reagent concentration a plateau of residual activity was reached after some time; its value was inversely proportional to TosL ysCHCl concentration but the plateau was not due to inactivator deplet ion. On the other hand, in the presence of Ce2+, phosphatidylserine an d phorbol 12-myristate 13-acetate, the activity loss followed saturati on kinetics, with k(inact) = 0.6 x 10(-3) s-1 and K(inact) = 1.9 mM. T he study of protection effects by ATP Mg2+ and histone required the pr esence of 50% glycerol in the incubation mixtures, otherwise the contr ols (kinase in the presence of activators and ATP Mg2+ or histone) rap idly 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(i nact) = 0.2 mM); ATP Mg2+ proved to afford a mixed competitive-non com petitive protection effect, while histone protected in a competitive m anner with a K(p) of 0.06 mug/ml. In the presence and the absence of g lycerol, plots of log k(obs) versus log inactivator concentration yiel ded straight lines with slopes of 0.7-0.9, indicating that 1 mol of re agent is sufficient for inactivation. The results described in this pa per suggest that the reagent TosLysCH2Cl hits die catalytic domain of activated PKC at the active site, which is not available in resting PK C; in non-activated PKC, the labeling site would be different.