Molecular weight of Na, K-ATPase approximated by the radiation inactivation method

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Abstract

The Na, K-ATPase has not yet been purified as a single molecular species, mainly because of its insolubility. Nevertheless, an approximate molecular weight of the dried enzyme can be obtained by the radiation inactivation technique introduced by Hutchinson and Pollard (1961). Using this procedure Kepner and Macey (1966) reported a value of about one million as the molecular weight for the ATPase of freeze-dried human erythrocyte ghosts. Their preparation contained both the ouabain sensitive and insensitive ATPase. A reinvestigation of this problem seemed worthwhile in that our preparation of Na, K-ATPase from brain microsomes is highly specific, essentially free of the ouabain-insensitive enzyme (Nakao et al., 1965). This preparation showed that the phosphorylated protein, obtained in a reaction with ATP32 requiring both Na and Mg ions, consisted of a single chemical species as indicated by the time course of hydrolysis and by its sensitivity towards hydroxylamine. In this report we have estimated the target molecular weight of the enzyme involved in the phosphorylation reaction with ATP and in the overall hydrolysis of ATP.

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