Biochemical Studies on <H^+> -ATPase and <Ca^(2+)> / <H^+> antiporter on the Yeast Vacuolar Membranes

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(1) Subunit a (Mr=89K) of the vacuolar membrane <H^+> -ATPase of the yeast S. cerevisiae was found to bind 8-azide-[ <(alpha)-^(32)> ]ATP. Labeling by this photo-sensitive ATP derivative was saturable with an apparent dissociation constant of <10^(-6)> M to <10^(-5)> M and decreased in the presence of ATP and ADP.(2) The enzyme was inactivated by NBD-Cl, about 1 <micro> M causing half maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or AMP-PNP. The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with [ <^(14)C> ]NBD-Cl, subunit a was specifically labeled and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. It was concluded that subunit a of the yeast vacuolar … More <H^+> -ATPase has a catalytic site that contains a single, essential tyrosine residue.(3) The kinetics of non-steady state hydrolysis of [ <gamma> - <^(32)P> ]ATP indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site. NBD-Cl inactivated the single catalytic site and inhibited the formation of an enzyme-ATP complex. A reaction mechanism for ATP hydrolysis with catalytic site cooperativity by the yeast vacuolar <H^+> -translocating ATPase was proposed.(4) Monoclonal antibodies against the vacuolar membrane and purified vacuolar membrane ATPase were systematically obtained. Now, we started to isolate genes of various vacuolar membrane proteins.(5) To elucidate the mechanism for generating the electrochemical potential difference across the vacuolar membrane, we analyzed the conductivities of vacuolar membrane for various ions. We found a novel <Cl^-> transport system and potential dependent ion channel on the vacuolar membrane. Less