Role of Multiple Phosphorylation in Monovalent Cation Transport ATPases

猪胃H/K-ATP酶α链Tyr ^7和Tyr ^4的可逆磷酸化作用最初在大鼠、家兔和大鼠体内得到证实。<10>这些数据和其他数据还表明，在顶端膜中存在一些重要的酶系统，它们与H/K-ATP酶α链上的Tyr ^7、Tyr ^7和Se ^7残基的可逆磷酸化作用距离很近。<10><27>酪氨酸激酶被抗c-Src抗体识别。丝氨酸激酶被PKC α和PKC β II抗体识别。蛋白磷酸酶-1的存在下，也进行了免疫学检测。用CHAPS溶解的G_1膜和其它膜进行柱层析分离，结果表明Src-激酶的表观分子量约为60kDa，PKC α和/或PKC β Ⅱ的表观分子量约为80kDa，Tyr-磷酸酶的表观分子量约为200kDs，PP-1的表观分子量约为35kDs，[γ-^P] ATP对猪胃H/K-ATP酶的磷酸化中间体（E ^P）/mol α链的最大量约为0.5，为^Pi的一半。<32><32><32>在有[γ-^P] ATP存在的情况下，在转换过程中酶与^P的最大结合是由于0.5 mol的E ^P +0.5 mol的酸不稳定酶结合的[γ-^P] ATP（EATP）。<32><32><32><32>H ^+-ATP酶活性/（EP + EATP）非常接近EP分解和Pi释放的表观速率常数。随着ATP浓度的增加，释放的Pi量与消失的EP量之比从1.01增加到1.02。这代表了第一个直接证据，对于P型ATP酶的情况，其中在涉及串扰的ATP水解过程中，对于一半的酶分子，从1 mol EP和对于另一半的1 mol EATP同时释放2 mol Pi。

Reversible phosphorylations of Tyr^7 and Tyr^<10> of pig stomach H/K-ATPase α-chain were initially demonstrated in vivo in rat, rabbit and. These data and others also suggest that some important enzyme systems are present in the apical membrane and in sufficiently proximity to participate in the reversible phosphorylation of Tyr^7 and Tyr^<10> and Se^<27> residues of the α-chain of H/K-ATPase. The tyr-kinase is recognized by anti-c-Src antibody. The Ser-kinase is recognized by antibodies against PKCα and PKC βII. The presence of protein phosphatase-1 was also immunologically detected. Column chromatographic separation of CHAPS solubilized G1 membrance and others indicate the appearance molecular weight of the Src-kinase to be 〜60 kDa, the PKCα and/or PKCβII to be 80 kDa, the Tyr-phosphatase to be 200 kDs and PP-1 to be 〜35 kDs.The maximum amount of phosphorylated intermediate (E^<32>P) / mol of α-chain of pig stomach H/K-ATPase from [γ-^<32>P]ATP was found to be 〜0.5, which was half of that formed from ^<32>Pi. The maximum ^<32>P binding for the enzume during turnover in the presence of [γ-^<32>P]ATP was due to 0.5 mol of E^<32>P + 0.5 mol of and acid labile enzyme bound [γ-^<32>P]ATP (EATP). The H^+ -ATPase activity/(EP + EATP), was very close to the apparent rate constants for EP breakdown and Pi liberation. The ratio of the amount of Pi liberated to that of EP that disappeared, increased from 1to 〜 2 with increasing concentrations of ATP. This represents the first direct evidence, for the case of a P-type ATPase in which 2 mol of Pi liberation occurs simultaneously from 1 mol of EP for half of he enzyme molecules and 1 mol of EATP for the other half, during ATP hydrolysis involving in crosstalk.