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

(1)酿酒酵母液泡膜&lt；H^+&gt；-ATPase的a亚基(mR=89K)与8-叠氮-[&lt；(α)-^(32)&gt；]ATP结合。这种光敏的ATP衍生物的标记是饱和的，表观解离常数为~lt；10^(-5)&gt；M，在ATP和ADP存在下降低。(2)该酶被NBD-Cl失活，在中性pH范围内约1&lt；micro&gt；M造成一半最大失活。这种失活可被ATP、ADP或AMP-PNP的存在所阻止。用2-巯基乙醇处理失活的酶，使其恢复原来的活力。对失活的动力学和化学研究表明，对每个酶分子中的一个酪氨酸残基进行化学修饰后，该酶失去了活性。当酶被[&lt；^(14)C&gt；]NBD-Cl灭活时，a亚基被特异性标记，这种标记可被ATP、GTP、ADP或AMP-PNP的存在完全阻止。结论：酵母液泡状…的a亚基More&lt；H^+&gt；-ATPase有一个含有单一必需酪氨酸残基的催化中心。(3)[&lt；Gamma&gt；-&lt；^(32)P&gt；]ATP的非稳态水解动力学表明，酶-ATP复合体的形成以及随后结合的ATP在NBD-Cl敏感的催化中心被水解为ADP和PI。NBD-Cl使单一催化位失活，并抑制酶-ATP复合体的形成。提出了酵母液泡膜-H~+-ATPase与催化位协同作用的反应机理。(4)系统地获得了抗液泡膜ATPase的单抗和纯化的液泡膜ATPase。现在，我们开始分离各种液泡膜蛋白的基因。(5)为了阐明产生跨液泡膜电化学电位差的机制，我们分析了液泡膜对各种离子的电导。我们在液泡膜上发现了一种新的~lt；Cl~-&gt；转运系统和电位依赖性离子通道。较少

(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