KINETIC-ANALYSIS OF THE INTRACELLULAR CONJUGATION OF MONOCHLOROBIMANE BY IC-21 MURINE MACROPHAGE GLUTATHIONE-S-TRANSFERASE

Monochlorobimane (MCB) reacts with glutathione (GSH) in a reaction catalyzed by the glutathione-S-transferase (GST) isozymes. The diffusion of MCB through cell membranes is rapid and the fluorescent conjugates are relatively insensitive to quenching and to pH effects, and are expelled slowly from the cell, allowing the rate of fluorescence increase to be used to probe the dynamics of the intracellular reaction. Using low-light microscopic cytometry to monitor the initial rates of fluorescence increase for the GST-catalyzed reaction within IC-21 macrophages yields V-max = 8.4.10(-16) mol s(-1) cell(-1) and K-m(MCB) = 65 mu M. Combining these data with an integrated Michaelis analysis of the reaction course yields K-I(P) approximate to 1.5.10(-5) M, and K-m(GSH) approximate to 3.0.10(-4) M (at [MCB] = 50 mu M). The values of V-max and K-m(MCB) for the cell-free (extracellular) GST-catalyzed conjugation reaction are 1.2 10(-18) mol s(-1) cell(-1) and 3.1 mu M, respectively. The values of V-max for the intra- and extracellular conjugation reactions differ by 700-fold, suggesting the presence of an intracellular activator for this enzyme system.