Optimal conditions for measurement of Na+,K+-ATPase activity of human leucocytes.
The Na+,K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. 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 degrees C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 mumol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at -20 degrees C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. The leucocyte Na+,K+-ATPase enzyme activity in healthy subjects was 186 mumol of Pi h-1g-1 of protein (median) with a range 136-243 mumol of Pi h-1g-1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.
Baron
D N
DN
Khan
F A
FA
D002021:Buffers; D004492:Edetic Acid; D000431:Ethanol; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology*; D009994:Osmolar Concentration; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood*; D013048:Specimen Handling / Q000379:methods; D013696:Temperature; D013997:Time Factors
D002021:Buffers; D000431:Ethanol; D010042:Ouabain; D004492:Edetic Acid; D000254:Sodium-Potassium-Exchanging ATPase