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.

最初在大鼠，兔和大鼠，兔子和体内证明了Tyr^7和Tyr^<10>的可逆磷酸化。这些数据和其他数据还表明，根尖膜中存在一些重要的酶系统，并且具有足够的接近性，可以参与Tyr^7和Tyr^<10>的可逆磷酸化以及H/K-ATPase的α-链的残基。抗C-SRC抗体识别Tyr-激酶。 Ser-激酶通过针对PKCα和PKCβII的抗体识别。还检测到蛋白质磷酸酶-1的存在。 Chaps溶剂G1膜和其他的柱色谱分离表明，SRC-激酶的外观分子量为〜60 kDa，PKCα和 /或PKCβII为80 kDa为80 kDa，Tyr-磷酸酶为200 kds and pp-1，pp-1为〜35 kds。发现来自[γ- ^<32> p] ATP的猪胃H/k-atpase为〜0.5，是由 ^<32> p形成的一半。在[γ-^<32> p] ATP存在下，在周转期间，ENZUME的最大^<32> P结合是由于0.5 mol e^<32> p + 0.5 mol 0.5 mol的和酸标记酶结合[γ-^<32> p] atp（eatp）。 H^ + -ATPase活性/（EP + EATP）非常接近于EP分解和PI解放的明显速率常数。随着ATP浓度升高，PI释放的PI量与EP消失的比率增加到2。这代表了第一个直接证据，对于P型ATPase的情况下，在ATP水解涉及Crosstalk的ATP水解期间，在另一半中，只有1摩尔EP发生2摩尔的Pi释放，而另一半毫克的EATP。