Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.
Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.
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具有接近最大比活性和磷酸化能力的Na,K-ATP酶的制备：反应机制涉及所有位点的证据。
DOI：
10.1021/bi983019b
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Sachs,JR
中科院分区：
文献类型：
--
作者：
Martin,DW;Sachs,JR
常用的Na+，K+- atp酶制剂的磷酸化能力远远低于从cDNA序列推断出的酶的分子量。这导致了酶反应机制的半位点或翻转模型的流行。我们从盐适应鸭的鼻盐腺中制备了Na+，K+- atp酶，其磷酸化能力和比活性接近理论最大值。37°C下比活性为>60 μmol （mg蛋白）-1min- 1min-1的制剂，磷酸化>60 nmol/mg蛋白，酶的周转速率为9690 min-1，在其他来源的酶报道的范围内。该酶的最大比活性分数与SDS - PAGE上α和β链的蛋白质分数比较好，特别是在最高比活性时，表明所有αβ原聚物都是有活性的。活性最强的制剂的凝胶只含有α和β链，而活性较弱的制剂含有许多外来蛋白质。主要的污染物是肌动蛋白。该制剂不含任何在γ亚基分子量范围内迁移的蛋白质。酶的亚基组成为α1和β1。这是首次报道一种纯、均质、完全活性的蛋白质制备方法。包含半位点或翻转机制的反应模型不适用于该酶。
The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 μmol (mg of protein)-1min-1at 37 °C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS−PAGE which was α and β chains, especially at the highest specific activity which indicates that all of the αβ protomers are active. The gels of the most reactive preparations contained only α and β chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the γ subunit. The subunit composition of the enzyme was α1and β1only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.