Elsevier

Analytical Biochemistry

Volume 258, Issue 1, 10 April 1998, Pages 63-67
Analytical Biochemistry

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

https://doi.org/10.1006/abio.1998.2572Get rights and content

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 μM, 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−1wet wt (1745 ± 84 pmol min−1· mg−1protein). 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.

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    This research was supported by a grant from Victoria University of Technology. S.F. was supported by an Australian Postgraduate Research Award.

    ☆☆

    Hargreaves, M.Thompson, M. W.

    2

    To whom correspondence should be addressed. E-mail:[email protected].

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