Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity.
Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods
D010042:Ouabain; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium