Purification and properties of a highly active ouabain-sensitive Na+, K+-dependent adenosinetriphosphatase from cardiac tissue

Abstract

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 K_m 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 K_i'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.