Studies of Oligomer Interaction in Cation Pumps in Real Time

用荧光探针(BIPM)研究了猪肾脏Na，K-ATPase中E1P向EP2转化过程中Na~+的转运。在BIPM标记的Na，K-ATPase中加入10um的三磷酸腺苷使其荧光强度增加，表观速率为137/S，磷酸化表观速率为119/S.这些结果表明，BIPM信号和磷酸酶的形成具有相似的动力学，因此，E1P转化为E2P的速度很快.在生理[ATP]条件下，E2P释放Na+的速率受到磷酸化和/或Na+解锁构象转变的限制。当猪胃膜H^+，K^+-ATPase制剂与[Gamma-^&lt；32&gt；P]ATP、Mg^&lt；2+&gt；和Ca^&lt；2+&gt；，32&gt；P结合到H^+，K^+-ATPase的Alphachain中的酪氨酸和丝氨酸残基中。并入的放射性物质被证明是翻转的。温和的对甲苯基苯丙氨基氯甲基酮-胰酶经反相柱层析处理后得到…更多的REE放射性活性多肽峰。第一个峰和第二个峰分别被指定为相同的氨基末端磷酸肽，包括Boty Tyr^&lt；10&gt；(^&lt；32&gt；P)和Tyr^7(^&lt；32&gt；P)和Tyr^&lt；10&gt；(^&lt；32&gt；P)，这两个峰也是通过胰酶处理与[Gamma-^&lt；32&gt；P]ATP，mg^&lt；2+&gt；和钒(Togawa K.，Ishiguro T.，Kaya S.，Shimada A.，Imagawa T.和Taniguchi K.(1995)J.Biol.)化学。270、15475-15478)。用胰酶处理部分纯化的H^+，K^+-ATPase制剂，与[Gamma-^&lt；32&gt；P]ATP，mg；&lt；2+&gt；和蛋白激酶-C或蛋白激酶-A孵育后，也可获得第三个峰。每三个峰组分中加入内切酶ASp-N，可得到一个主要的^&lt；32&gt；P峰，表明为Asp^&lt；21&gt；-Met-Ala-Lys-Met-Ser(^&lt；32&gt；P)-Lys-Lys-Lys^&lt；。30；在阿尔法链中。这些数据和其他数据表明，含有Tyr^7、Tyr^&lt；10&gt；和Ser^&lt；27&gt；的H^+，K^+-ATPaseα链的氨基末端结构域是蛋白激酶依赖的磷酸化的热点。数据还表明，钙离子和cAMP参与了这些磷酸化反应。较少

The fluorescent probe, (BIPM), was used to monitor Na^+ translocation coupled to the conversion of E1P to EP2 in pig kidney Na, K-ATPase. The addition of 10 muM ATP to BIPM-labeled Na, K-ATPase gave an increase in fluorescence intensity with an apparent rate of 137/s and the phosphorylation with an apparent rate of 119/s. These results show that the BIPM signal and phosphoenzyme formation have similar kinetics and that, consequently, the conversion of E1P to E2P is very fast. At physiological[ATP], Na^+ release from E2P is rate-limited by phosphorylation and/or the conformational transitions involving Na^+ deocclusion.When pig stomach membrane H^+, K^+-ATPase preparations were incubated with[gamma-^<32>P]ATP,Mg^<2+> and Ca^<2+>, ^<32>P were incorporated into Tyr and Ser residues in the alphachain of H^+, K^+-ATPase. The radioactivity incorporated was shown to turn over. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatmentifollwed by a reverse-phase column chromatography gave th … More ree radio active peptide peaks. The first and the second peaks were assigned, respectively, to be the same amino-terminal phospho peptides, containing boty Tyr^<10>(^<32>P) and Tyr^7(^<32>P) and Tyr^<10>(^<32>P), both of which had been also obtained by the trypsin treatment of the preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and vanadate (Togawa K., Ishiguro T., Kaya S., Shimada A., Imagawa T., and Taniguchi K.(1995)J.Biol. Chem. 270,15475-15478). The third peak was also obtained after the trypsin treatement of partially purified H^+, K^+-ATPase preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and protein kinase-C + Ca^<2+> or protein kinase-A.Addition of endoproteinase ASP-N to each third peak fraction gave a major ^<32>P peptide peak as shown to be Asp^<21>-Met-Ala-Lys-Met-Ser(^<32>P)-Lys-Lys-Lys^<30> in the alpha-chain. These data and others indicate that amino-terminal domain of the alpha-chain of H^+, K^+-ATPase containing Tyr^7, Tyr^<10> and Ser^<27> is a hot spot for protein kinases dependent phosphorylation. The data also suggest participation of Ca^<2+> and cAMP in these phosphorylation reactions. Less