Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.