Mg-dependent inhibition of beef heart soluble mitochondrial ATPase by tricyclic antipsychotics

The effect of fluphenazine and related phenothiazine and thioxanthene derivatives on beef heart soluble mitochondrial ATPase (EC 3.6.1.3) was studied under a precise control of the Mg2+-ATP equilibrium. These drugs were shown to be reversible, noncompetitive inhibitors with respect to the substrate (the Mg . ATP complex). The inhibition was found to be dependent on the concentration of free magnesium ions, although free Mg2+ was not essential for the interaction of the inhibitors with the enzymatic protein. Bicarbonate anions, which are known to antagonize the effect of free Mg2+ on the enzyme kinetics, also antagonized the drug-induced inhibition. Concentrations giving 50% inhibition of enzyme activity were in the micromolar range. Inhibitory potencies increased when the pH of the reaction mixture was lowered from 8.2 to 6.9. Cleland [The Enzymes (P. D. Boyer, ed.), Vol. II. Academic Press, New York, 1--65 (1970)] analysis of the inhibition, by means of slope and intercept replots, indicated that the inhibition was the result of the interaction with more than one drug molecule. All drugs tested afforded complete protection against the cold-induced inactivation of soluble mitochondrial ATPase. These results point to a specific mode of inhibition that mimics, in some respects, the action of the natural inhibitor protein of mitochondrial ATPase.