Proton adenosine triphosphatase complex of rat liver. The effect of trypsin on the F1 and F0 moieties of the enzyme.

The ATPase activity of rat liver Fl is inhibited by a number of covalent labeling agents which are reported to have preferential specificities for either tyrosine, carboxyl, or amino residues. Two types of agents, ie. trypsin and sulfhydryl reactive compounds, are without effect on the ATPase activity of F1. In this study, the effect of trypsin on energy-linked functions catalyzed by the mitochondrial ATPase complex (FoFI) of rat liver was studied under specified conditions (60 pg of trypsin/mg of F1 in 250 mM K P i , 10 mM CaC12, 5 mM EDTA, pH 7.5,20 min, 25°C). In reconstitution experiments, trypsin-treated F1 binds to F1-depleted urea particles in amounts equivalent to untreated F1. The reconstituted trypsin-treated F1 is equally as sensitive to inhibition by oligomycin and dicyclohexylcarbodiimide as the reconstituted untreated F1. However, the system reconstituted with trypsin-treated F1, unlike the system reconstituted with untreated F1, fails to catalyze ATP synthesis and ATPdependent transhydrogenase activity; it also fails to catalyze ATPand succinate-dependent proton translocation monitored with the fluorescent dye 9-amino-6chloro-2-methoxyacridine. The trypsin-treated F1 has velocity versus ATP kinetic profiles identical with the untreated F1 in both a nonactivating buffer, Tris-C1, and an activating buffer, Tris-HC03. The trypsin-treated F1 is equally as sensitive to product inhibition by ADP as the untreated enzyme. Moreover, trypsin has no effect on the amount of nucleotides bound to the isolated enzyme (1 mol of ATP and 1 mol of ADP/mol of F1), or the amount of added nucleotides which bind tightly to the enzyme (1 mol of adenyl-5”yl imidodiphosphate and 1 mol of ADP/mol of F1). However, two dimensional gel electrophoresis of trypsin-treated F1 reveals two new peptides comparable in size to the 6 subunit. If, rather than modifying F1 with trypsin, the Fl-depleted urea particles (containing F,) are treated with trypsin, similar results are obtained. That is, F, binds to the trypsin-treated urea particles in normal amounts, its ATPase activity is unaffected, and it is markedly inhibited by oligomycin and dicyclohexylcarbodiimide. However, the reconstituted system fails to participate in energy-linked functions. Trypsin has no effect on ATP-dependent proton translocation in inverted inner membrane vesicles or in urea particles after reconstitution with F1, under conditions identical with those which elicit effects on F1 or urea particles. * This work was supported by United States Public Health Service Grant CA10951 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. These results show that there are trypsin-sensitive regions both on F1 and on the membrane (most likely at the level of Fo) which are essential for transformation of the electrochemical gradient of protons into chemical energy (and vice versa). Because these sites are inaccessible to trypsin in inverted inner membrane vesicles and in urea particles reconstituted with F1, they most likely lie at the interface between the Fo and F1 moieties of the FoF1.ATPase complex. The results also suggest that the mode of action of oligomycin and dicyclohexylcarbodiimide is either to elicit a conformational change in Fo which alters F1 or to block proton flow through one of two possible channels in the Fo unit.