Role of Multiple Phosphorylation in Monovalent Cation Transport ATPases

在大鼠、家兔和猪胃H/ k - atp酶α-链Tyr^7和Tyr^<10 b>的体内初步证实了可逆磷酸化。这些数据和其他数据还表明，一些重要的酶系统存在于顶膜中，并且距离足够近，可以参与H/ k - atp酶α-链上Tyr^7和Tyr^<10>和Se^<27>残基的可逆磷酸化。酪氨酸激酶被抗c- src抗体识别。ser -激酶可被PKCα和PKC βII抗体识别。蛋白磷酸酶-1的存在也被免疫检测。CHAPS溶解G1膜等的柱层析表明，src激酶的外观分子量为~ 60 kDa， PKCα和/或PKCβII为80 kDa，酪氨酸磷酸酶为200 kDs， PP-1为~ 35 kDs。[γ-^<32>P]ATP对猪胃H/ k -ATP酶α-链的最大磷酸化中间体(E^<32>P) / mol的最大磷酸化量为~ 0.5，是^<32>Pi的一半。在[γ-^<32>P]ATP存在的情况下，酶在周转过程中最大的^<32>P结合是由于0.5 mol E^<32>P + 0.5 mol酸不稳定酶结合[γ-^<32>P]ATP （EATP）。H^+ - atp酶活性/(EP + EATP)与EP分解和Pi释放的表观速率常数非常接近。随着ATP浓度的增加，Pi的释放量与EP的消失量之比从1增加到~ 2。这是第一个直接证据，证明在涉及串扰的ATP水解过程中，p型ATP酶的一半分子在1 mol EP和另一半分子在1 mol EATP的作用下同时发生2 mol Pi释放。

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.