Optimal Conditions for Measurement of Na+, K+-ATPase Activity of Human Leucocytes

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Abstract
1. The Na+, K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. 2. The maximum enzyme activity was achieved under the following conditions: concentration (mmol/l), Tris/HCl 50, Na 100, K 15, ATP 5, Mg 7, EDTA 1; pH 7.2 and temperature 37°C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 μmol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. 3. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at −20°C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. 4. The leucocyte Na+, K+-ATPase enzyme activity in healthy subjects was 186 μmol of Pi h−1 g−1 of protein (median) with a range 136-243 μmol of Pi h−1 g−1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.