title: Measurement of Na+, K+-ATPase activity in human skeletal muscle.
abstract: There are few published measures of Na+,K+-ATPase activity in human skeletal muscle. This study investigated the suitability of the K+-stimulated 3-O-methylfluorescein phosphatase assay for measurement of Na+,K+-ATPase activity in human skeletal muscle. Factors investigated include enzyme kinetics, sample treatment, and ligand concentration. The addition of ouabain blocked maximal K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, confirming the specificity of the assay. Activity was maximal using a multiple freeze-thaw treatment of the homogenate, a 10 mM KCl activating concentration, and a 3-O-methylfluorescein phosphatase substrate concentration of 160 microM, which is eight times higher than previously reported. From quadriceps muscle biopsies taken from seven healthy untrained subjects, the maximal K+-stimulated 3-O-MFPase activity determined from the homogenates was (mean +/- SE) 292 +/- 10 nmol min-1 . g-1 wet wt (1745 +/- 84 pmol min-1 . mg-1 protein). This value is five times greater than previously published data for human skeletal muscle. The intra-assay variability was 8.1% and the interassay variability was 5.3%. These modifications greatly enhanced the 3-O-MFPase assay, with the improved enzymatic conditions allowing valid, reliable measurement of Na+,K+-ATPase activity in small samples of human skeletal muscle.
authors.affiliation: Centre for Rehabilitation, Exercise, and Sport Science, Victoria University of Technology, Melbourne, Victoria, 8001, Australia.
authors.forename: S F
authors.initials: SF
authors.lastname: Fraser
authors.forename: M J
authors.initials: MJ
authors.lastname: McKenna
mesh_terms: D000328:Adult; D006801:Humans; D007700:Kinetics; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: C045349:3-O-methyl-fluorescein phosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium