Larsen J S, Kjeldsen K
Department of Medicine B2142, Rigshospitalet University of Copenhagen School of Medicine, Denmark.
Basic Res Cardiol. 1995 Jul-Aug;90(4):323-31. doi: 10.1007/BF00797910.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
通过测定钾离子和钙离子依赖性对硝基苯磷酸酶（pNPPase）活性，对大鼠心室肌粗匀浆中钠钾ATP酶和钙ATP酶进行完全定量的分析方法得到了评估和优化。使用这些分析方法，发现11至12周龄大鼠心室肌中总的钾离子和钙离子依赖性pNPPase活性分别为2.98±0.10和0.29±0.02μmol·min⁻¹·g⁻¹湿重（平均值±标准误）（n = 5）。个体间测定的变异系数分别为7%和12%。总的钠钾ATP酶和钙ATP酶浓度估计分别为2和10nmol·g⁻¹湿重。对假定的发育变化进行评估发现，大鼠心肌中钙离子依赖性pNPPase活性呈现双相年龄相关变化，从出生到生命第三周左右增加，随后下降。相比之下，大鼠心肌中钾离子依赖性pNPPase活性从出生到成年呈下降趋势。排除了这些变化是心肌水和蛋白质含量变化的简单结果。以每颗心脏表示，钾离子和钙离子依赖性pNPPase活性逐渐增加至平稳状态。目前用于钠钾ATP酶定量的分析方法相对于[³H]哇巴因结合法具有优势，可应用于对哇巴因耐药的大鼠心肌，并且比在粗组织匀浆中测量钾离子依赖性3 - O - 甲基荧光素磷酸酶（3 - O - MFPase）更简单、快速。此外，只需少量修改，pNPPase分析就能对粗心肌匀浆中的钙ATP酶进行定量。（摘要截断于250字）
