Quantification in crude homogenates of rat myocardial Na+, K(+)- and Ca(2+)-ATPase by K+ and Ca(2+)-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na+, K(+)- and Ca(2+)-ATPase in crude homogenates of rat ventricular myocardium by determination of K(+)- and Ca(2+)-dependent p-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K(+)- and Ca(2+)-dependent pNPPase activities in ventricular myocardium of 11-12 week-old rats were found to be 2.98 +/- 0.10 and 0.29 +/- 0.02 mumol x min-1 x g-1 wet wt. (mean +/- SEM) (n = 5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na+, K(+)- and Ca(2+)-ATPase concentrations were estimated to 2 and 10 nmol x g-1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca(2+)-dependent pNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K(+)-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple outcome of variations in water and protein content of myocardium. Expressed per heart, the K(+)- and Ca(2+)-dependent pNPPase activity gradually increased to a plateau. The present assay for Na+, K(+)-ATPase quantification has the advantage over [3H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) in crude tissue homogenates. Furthermore, with few modifications the pNPPase assay allows quantification of Ca(2+)-ATPase on crude myocardial homogenates.