心脏组织中高活性瓦阿因敏感Na+, K+依赖性腺苷三磷酸酶的纯化和性质

Hideo Matsui, Arnold Schwartz
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引用次数: 213

摘要

通过脱氧胆酸盐和NaI连续处理,从小心肌中获得了一种高度活性和特异性的Na+, K+依赖性atp酶。研究了酶的一般性质。同时添加Na+和K+使其活性比基本依赖Mg2+的水平提高了约20倍。10−4 M的瓦巴因完全逆转了活化。最适pH为7.2;Na+依赖性、K+依赖性和Mg2+依赖性酶活性的ATP Km分别为2.4·10−4 M和5.0·10−5 M。只有CTP可以取代ATP,但活性是ATP的14%。叠氮化物和组蛋白对粗ATPase系统有影响,对纯化酶无影响。对氯甲苯甲酸盐、n -乙基马来酰亚胺、寡霉素、三丁基氯化锡、辛基胍和乌阿巴因对Na+、K+依赖性成分有抑制作用,而对碱性Mg2+依赖性活性几乎没有影响。Ki值分别为5·10−6 M、6·10−4 M、7·10−6 M、1.5·10−5 M、3·10−4 M和10−6 M。
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Purification and properties of a highly active ouabain-sensitive Na+, K+-dependent adenosinetriphosphatase from cardiac tissue

A highly active and specific Na+, K+-dependent ATPase was obtained from calf-heart muscle by succesive treatments with deoxycholate and NaI.

General properties of the enzyme were studied. Simultaneous addition of Na+ and K+ increased the activity about 20 times above the basic Mg2+-dependent level. The activation was completely reversed by 10−4 M ouabain. The optimal pH was 7.2; the Km for ATP for the Na+,K+-dependent and Mg2+-dependent enzyme activities were 2.4·10−4 M and 5.0·10−5 M, respectively. Only CTP could be substituted for ATP, but the activity was 14% of that found with ATP.

Azide and histone, which affected the crude ATPase system, had no effect on the purified enzyme. p-Chloromercuribenzoate, N-ethylmaleimide, oligomycin, tributyltin chloride, octylguanidine as well as ouabain were found to inhibit the Na+, K+-dependent component while having little or no effect on the basic Mg2+-dependent activity. The Ki's were 5·10−6 M, 6·10−4 M, 7·10−6 M, 1.5·10−5 M, 3·10−4 M and 10−6 M, respectively.

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