David P, Horne W C, Baron R
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
Biochim Biophys Acta. 1996 Apr 3;1280(1):155-60. doi: 10.1016/0005-2736(95)00284-7.
Recent reports indicate that vacuolar-type proton ATPases from chicken osteoclasts (Chatterjee et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6257-6261), yeast vacuoles and chromaffin granules (Beltran and Nelson (1992) Acta Physiol. Scand. Suppl. 607, 41-47) can be inhibited by vanadate, albeit at a concentration much higher than that required to inhibit P-type ATPases. We have characterized the mechanism by which vanadate inhibits vacuolar-type ATPase-mediated proton transport by chicken kidney microsomes. The initial rate of proton transport is somewhat less sensitive to vanadate than the total acidification, with IC50 values of 1.58 mM and 0.78 mM vanadate, respectively. The inhibition of both the initial rate and total acidification is noncompetitive with respect to ATP. The inhibition is abolished when ADP is removed by an ATP-regenerating system, and the addition of exogenous ADP increases the vanadate inhibition of proton transport in a synergistic manner, thus demonstrating that inhibition by vanadate is dependent on the presence of ADP and explaining the lower effect of vanadate on the initial rate of acidification. Phosphate protects proton transport activity from inhibition by vanadate. These effects of ADP and phosphate suggest that inhibition by vanadate may involve the formation of a complex with ADP at a nucleotide binding site, possibly at the catalytic site of the enzyme.
最近的报告表明，鸡破骨细胞（Chatterjee等人，1992年，《美国国家科学院院刊》89卷，6257 - 6261页）、酵母液泡和嗜铬颗粒（Beltran和Nelson，1992年，《生理学杂志增刊》607卷，41 - 47页）中的液泡型质子ATP酶可被钒酸盐抑制，尽管所需浓度远高于抑制P型ATP酶的浓度。我们已对钒酸盐抑制鸡肾微粒体中液泡型ATP酶介导的质子转运的机制进行了表征。质子转运的初始速率对钒酸盐的敏感性略低于总酸化作用，钒酸盐的IC50值分别为1.58 mM和0.78 mM。初始速率和总酸化作用的抑制对ATP均无竞争性。当通过ATP再生系统去除ADP时，抑制作用消失，添加外源ADP会以协同方式增强钒酸盐对质子转运的抑制作用，从而表明钒酸盐的抑制作用取决于ADP的存在，并解释了钒酸盐对酸化初始速率影响较小的原因。磷酸盐可保护质子转运活性免受钒酸盐的抑制。ADP和磷酸盐的这些作用表明，钒酸盐的抑制作用可能涉及在核苷酸结合位点（可能是酶的催化位点）与ADP形成复合物。
