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Measurement of Na+, K+-ATPase activity in human skeletal muscle

journal contribution
posted on 10.04.1998, 00:00 authored by Steve FraserSteve Fraser, M J McKenna
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 biochemistry

Volume

258

Issue

1

Pagination

63 - 67

Publisher

Elsevier

Location

Amsterdam, The Netherlands

ISSN

0003-2697

Language

eng

Publication classification

C1.1 Refereed article in a scholarly journal

Copyright notice

1998, Academic Press