Measurement of Na+,K+-ATPase Activity in Human Skeletal Muscle

✍ Scribed by Steve F. Fraser; Michael J. McKenna

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 96 KB
• Volume: 258
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.2572

✦ Synopsis

There are few published measures of Na ؉ ,K ؉ -AT-Pase activity in human skeletal muscle. This study investigated the suitability of the K ؉ -stimulated 3-Omethylfluorescein 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-Omethylfluorescein 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 M, 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 ⅐ g ؊1 wet wt (1745 ؎ 84 pmol min ؊1 ⅐ 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.