Pressure effects on the interaction between natural inhibitor protein and mitochondrial F1-ATPase.

Pressure stability of the complex formed between F1-ATPase and the inhibitor protein (IP) was studied in the membrane-bound and soluble, purified forms of beef-heart mitochondrial enzymes. A latent preparation of submitochondrial particles (SMP-MgATP) initially exhibits low hydrolytic activity. Dissociation of IP increases the activity about 10-fold. This increase occurs in parallel with an increase in sensitivity to pressure inactivation. The membrane-bound, latent IP-F1-ATPase complex is activated 2.5-fold when incubated at a pressure of 1.7 kbar, suggesting dissociation of IP. A fully active preparation of submitochondrial particles depleted of IP (AS-particles) is highly pressure labile when compared with the latent form. In the absence of IP, soluble purified F1-ATPase is also inactivated by pressure. In contrast, the soluble IP-F1-ATPase complex is very resistant to pressure, as evidenced by enzymatic and fluorescence studies. Based on the pressure-titration experiments, binding of IP stabilizes the F1-ATPase complex by 1.54 kcal per mole of complex. The substrate MgATP confers additional protection on both preparations only in the presence of IP. Glycerol appears to prevent dissociation of IP and therefore protects SMP-MgATP from pressure inactivation. Our results demonstrate that in addition to its regulatory role in catalysis, IP stabilizes the structure of the F1-ATPase complex. The pressure-induced dissociation of IP from F1-ATPase and its prevention by glycerol suggest that nonpolar in addition to electrostatic interactions are important for the binding of IP to the regulatory site.