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Measurement of Na+, K+-ATPase activity in human skeletal muscle
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.
History
Journal
Analytical biochemistryVolume
258Issue
1Pagination
63 - 67Publisher
ElsevierLocation
Amsterdam, The NetherlandsPublisher DOI
ISSN
0003-2697Language
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
1998, Academic PressUsage metrics
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Categories
Keywords
AdultHumansKineticsMaleMuscle, SkeletalPotassiumReproducibility of ResultsSensitivity and SpecificitySodium-Potassium-Exchanging ATPaseSpectrometry, FluorescenceScience & TechnologyLife Sciences & BiomedicinePhysical SciencesBiochemical Research MethodsBiochemistry & Molecular BiologyChemistry, AnalyticalChemistryNA+-K+ PUMPSUBUNIT ISOFORMSQUANTIFICATIONTRANSPORTATPASE(NA++K+)-ATPASENUMBERCELLS