QUANTIFICATION IN CRUDE HOMOGENATES OF RAT MYOCARDIAL NA-ATPASE AND CA2+-ATPASE BY K+-DEPENDENT AND CA2+-DEPENDENT PNPPASE - AGE-DEPENDENT CHANGES(,K+)

Assays for complete quantification of Na+, K+- and Ca2+-ATPase in crud e homogenates of rat ventricular myocardium by determination of K+- an d Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activities were e valuated and optimized. Using these assays the total K+- and Ca2+-depe ndent pNPPase activities in ventricular myocardium of 11-12 week-old r ats were found to be 2.98 +/- 0.10 and 0.29 +/- 0.02 mu mol x min(-1) x g(-1) wet wt. (mean +/- SEM) (n = 5), respectively. Coefficient of v ariance of interindividual determinations was 7 and 12 %, respectively . The total Na+, K+- and Ca2+-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 Ca2+-dependent pNPPase activity with an increase from b irth to around the third week of life followed by a decrease. By contr ast, the K+-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes wer e simple outcome of variations in water and protein content of myocard ium. Expressed per heart, the K+- and Ca2+ dependent pNPPase activity gradually increased to a plateau. The present assay for Na+, K+-ATPase quantification has the advantage over [H-3] 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 pho sphatase (3-O-MFPase) in crude tissue homogenates. Furthermore, with f ew modifications the pNPPase assay allows quantification of Ca2+ ATPas e on crude myocardial homogenates. Age-dependent changes in K+- and Ca 2+-dependent pNPPase activities are of developmental interest and indi cate the importance of close age match in studies of quantitative aspe cts of Na+, K+- and Ca2+-ATPase in excitable tissues.