An improved procedure for the preparation and measurement of (Na + + K +)-ATPase in human erythrocytes

Abstract

Sodium and potassium-stimulated adenosine triphosphatase ((Na + + K +)-ATPase) activity and membrane permeability as measured by 22Na influx were compared in erythrocyte membranes prepared by hypotonic and detergent (ionic and non-ionic) haemolysis. The detergent-treated erythrocytes showed significantly ( P <0.001) greater (Na + + K +)-ATPase and permeability to 22Na compared with erythrocyte membranes prepared by hypotonic haemolysis. In addition, increased enzyme activities were exhibited when membranes initially prepared by hypotonic haemolysis were subsequently exposed to 1% (w/v) saponin in the reaction mixture. A maximum level of (Na + + K +)-ATPase was achieved with 1.5 mg/ml sodium deoxycholate haemolysing agent. A method for erythrocyte vesicle preparation with sodium deoxycholate as haemolysing agent was developed. This method yielded high and reproducible (Na + + K +)-ATPase activity, which could be related to a new additional index of vesicle count. Biological variations and dietary habits influenced day to day levels of (Na + + K +)-ATPase activity but these fluctuations were kept to a minimum when blood samples were collected under identical conditions after an overnight fast, and a strictly standardized protocol of erythrocyte vesicle preparation was followed. Reproducibility of (Na + + K +)-ATPase activity from assay to assay could be predicted by monitoring the completeness of haemolysis, vesicle size-distribution graphs, vesicle count and vesicular protein concentrations. The initial packed erythrocyte volume and the enzyme assay volume yielded the most reliable indices of (Na + + K +)-ATPase activity, though vesicle count could also be used as an index.