Role of Multiple Phosphorylation in Monovalent Cation Transport ATPases

多重磷酸化在单价阳离子转运 ATP 酶中的作用

基本信息

批准号：
10308028
负责人：
TANIGUCHI Kazuya
金额：
$ 15.58万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10308028/
关键词：
Na K-ATpase H K-ATPase phosphorylation phosphatases kinase oligomer Hポンプフォスファターゼ

项目摘要

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.

猪胃H/K-ATP酶α-链的Tyr^7和Tyr^10的可逆磷酸化最初在大鼠、兔体内得到证实。这些数据和其他数据也表明一些重要的酶系统存在于顶端。膜并且足够接近，参与 H/K-ATPase α 链的 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。丝氨酸激酶被针对 PKCα 和 PKC βII 的抗体识别。CHAPS 溶解的 G1 膜的柱色谱分离也表明存在蛋白磷酸酶。 Src激酶的外观分子量为〜60 kDa，PKCα和/或PKCβII为80 kDa， Tyr-磷酸酶为200 kDs，PP-1为～35 kDs。磷酸化中间体(E^ 32 P)的最大量/来自[γ-的猪胃H/K-ATP酶的α-链的摩尔]发现^ 32 P]ATP为～0.5，这是由^ 32 Pi形成的ATP的一半。 ^ 32 P的最大结合。在存在[γ-^ 32> P]ATP的情况下，周转期间的enzume是由于0.5 mol E^ 32 P + 0.5 mol和酸不稳定酶结合的[γ-^ 32 P] ATP (EATP H^+ -ATPase 活性/(EP + EATP) 非常接近 EP 分解和 Pi 释放的表观速率常数。消失，随着 ATP 浓度的增加从 1 增加到〜 2 这代表了第一个直接证据，对于 P 型 ATP 酶的情况，其中一半的酶分子从 1 mol EP 中同时释放 2 mol Pi。另一半为1摩尔EATP，ATP水解过程中涉及串扰。