Peluffo R D, Garrahan P J, Rega A F
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Junín, Buenos Aires, Argentina.
J Biol Chem. 1992 Apr 5;267(10):6596-601.
Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.
在25℃以及一系列广泛的[τ-32P]ATP浓度下，对从猪肾外髓质纯化得到的钠钾ATP酶的预稳态磷酸化进行了研究。在10微摩尔ATP时，磷酸化遵循简单的指数动力学，40毫秒后达到稳定水平，即0.76±0.04纳摩尔磷/毫克蛋白质，kapp = 73.0±6.5秒-1。在500微摩尔ATP时，磷酸化的时间进程发生了巨大变化，因为磷酶以更高的速率（kapp≥370秒-1）达到了高两到四倍的水平，并在约40毫秒内降至与10微摩尔ATP时相同的稳定水平。在含有Mg2+、Na+和ATP的介质中进行周转的钠钾ATP酶中未观察到这种超磷酸化现象，这表明它需要酶处于静止状态。超磷酸化依赖于Mg2+和Na+，并被哇巴因和异硫氰酸荧光素完全抑制。变性后，超磷酸化产生的磷酶具有钠钾ATP酶α亚基的电泳迁移率，其水解被羟胺加速。在摩尔基础上，用1000微摩尔ATP磷酸化后，每结合一个哇巴因的磷酸盐化学计量比为2.40±0.60。这些结果与以下观点一致：在适当条件下，每个功能性钠钾ATP酶单位可以从ATP接受两个或更多个快速周转的磷酸盐。
