Bidwai A P, Morjana N A, Scarborough G A
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1989 Jul 15;264(20):11790-5.
The Neurospora crassa plasma membrane H+-ATPase is inactivated by the periodate-oxidized nucleotides, oATP, oADP, and oAMP, with oAMP the most effective. Inhibition of the ATPase is essentially irreversible, because Sephadex G-50 column chromatography of the oAMP-treated ATPase does not result in a reversal of the inhibition. Inhibition of the ATPase by oAMP is protected against by the H+-ATPase substrate ATP, the product ADP, and the competitive inhibitors TNP (2',3'-O-(2,4,6-trinitrocyclohexadienylidine)-ATP and TNP-ADP, suggesting that oAMP inhibition occurs at the nucleotide binding site of the enzyme. The rate of inactivation of the ATPase by oAMP is only slightly affected by EDTA, indicating that the oAMP interaction with the nucleotide binding site of the H+-ATPase occurs in the absence of a divalent cation. The protection against oAMP inhibition by ADP is likewise unaffected by EDTA. The inhibition of the ATPase by oAMP is absolutely dependent on the presence of acidic phospholipids or acidic lysophospholipids known to be required for H+-ATPase activity, suggesting that these lipids either aid in the formation of the nucleotide binding site or render it accessible. Incubation of the ATPase with Mg2+ plus vanadate, which locks the enzyme in a conformation resembling the transition state of the enzyme dephosphorylation reaction, completely protects against inhibition by oAMP, suggesting that in this transition state conformation the nucleotide site either does not exist, or is inaccessible to oAMP. Labeling studies with [14C] oAMP indicate that the incorporation of 1 mol of oAMP is sufficient to cause complete inactivation of the ATPase.
粗糙脉孢菌质膜H⁺-ATP酶可被高碘酸盐氧化的核苷酸oATP、oADP和oAMP失活，其中oAMP最有效。ATP酶的抑制作用基本上是不可逆的，因为用oAMP处理过的ATP酶经Sephadex G - 50柱层析后抑制作用并未逆转。oAMP对ATP酶的抑制作用可被H⁺-ATP酶底物ATP、产物ADP以及竞争性抑制剂TNP（2',3'-O-(2,4,6-三硝基环己二烯叉基)-ATP和TNP - ADP）所保护，这表明oAMP的抑制作用发生在该酶的核苷酸结合位点。oAMP使ATP酶失活的速率仅受到EDTA的轻微影响，这表明oAMP与H⁺-ATP酶核苷酸结合位点的相互作用是在没有二价阳离子的情况下发生的。ADP对oAMP抑制作用的保护同样不受EDTA影响。oAMP对ATP酶的抑制作用绝对依赖于已知H⁺-ATP酶活性所需的酸性磷脂或酸性溶血磷脂的存在，这表明这些脂质要么有助于核苷酸结合位点的形成，要么使其可被利用。将ATP酶与Mg²⁺加钒酸盐一起孵育，可使酶锁定在类似于酶去磷酸化反应过渡态的构象，从而完全防止oAMP的抑制作用，这表明在这种过渡态构象中，核苷酸位点要么不存在，要么oAMP无法接近。用[¹⁴C]oAMP进行的标记研究表明，掺入1摩尔oAMP足以导致ATP酶完全失活。
