Quinacrine mustard inactivates the bovine heart mitochondrial F1-ATPase with the modification of the beta subunit.

ABSTRACT

The bovine mitochondrial F1-ATPase is reversibly inhibited by quinacrine. The concentration of quinacrine which causes 50% inhibition at pH 7.0 is estimated to be 580 microM. Lineweaver-Burk plots constructed from kinetic data collected at pH 7.0 with equimolar concentrations of Mg2+ and ATP in the reaction mixtures indicate competitive inhibition by quinacrine. Uncompetitive inhibition by quinacrine is indicated by Lineweaver-Burk plots constructed from kinetic data obtained at pH 7.0 with variable ATP concentrations and a constant Mg2+ concentration of 3.0 mM. A KI of 440 microM was calculated from a replot of 1/Vmaxi versus quinacrine concentration using the intercepts of the Lineweaver-Burk plots constructed from the data obtained at a fixed concentration of 3.0 mM Mg2+. Quinacrine mustard is a potent inactivator of the F1-ATPase. The pseudo-first order rate constant, k1, for the inactivation of the enzyme by 500 microM quinacrine mustard is 0.161 min-1 at pH 7.0 and 23 degrees C. Under the same conditions in the presence of 5.0 mM ATP, 5.0 mM ADP, or 5.0 mM Mg2+ plus 5.0 mM ADP, k1 is, respectively 0.082 min-1, 0.136 min-1, or 0.075 min-1. In the presence of 1.0 mM chlorpromazine or 5.0 mM quinacrine under the above conditions, k1, is 0.089 min-1 and 0.037 min-1, respectively. Free Mg2+ has no effect on the rate of inactivation of the enzyme by quinacrine mustard. The rate of inactivation of the F1-ATPase by quinacrine mustard as a function of pH revealed an apparent pK alpha of 8.0. Examination of the resolved subunits for fluorescence after inactivating the enzyme with quinacrine mustard demonstrated that only the beta subunit was labeled.