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Measurement of Na+, K+-ATPase activity in human skeletal muscle.
Anal Biochem. 1998 Apr 10; 258(1):63-7.AB

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.

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Authors+Show Affiliations

Fraser SF
Centre for Rehabilitation, Exercise, and Sport Science, Victoria University of Technology, Melbourne, Victoria, 8001, Australia.
McKenna MJ
No affiliation info available

MeSH

AdultHumansKineticsMaleMuscle, SkeletalPotassiumReproducibility of ResultsSensitivity and SpecificitySodium-Potassium-Exchanging ATPaseSpectrometry, Fluorescence

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9527849

Citation

Fraser, S F., and M J. McKenna. "Measurement of Na+, K+-ATPase Activity in Human Skeletal Muscle." Analytical Biochemistry, vol. 258, no. 1, 1998, pp. 63-7.
Fraser SF, McKenna MJ. Measurement of Na+, K+-ATPase activity in human skeletal muscle. Anal Biochem. 1998;258(1):63-7.
Fraser, S. F., & McKenna, M. J. (1998). Measurement of Na+, K+-ATPase activity in human skeletal muscle. Analytical Biochemistry, 258(1), 63-7.
Fraser SF, McKenna MJ. Measurement of Na+, K+-ATPase Activity in Human Skeletal Muscle. Anal Biochem. 1998 Apr 10;258(1):63-7. PubMed PMID: 9527849.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Measurement of Na+, K+-ATPase activity in human skeletal muscle. AU - Fraser,S F, AU - McKenna,M J, PY - 1998/5/2/pubmed PY - 1998/5/2/medline PY - 1998/5/2/entrez SP - 63 EP - 7 JF - Analytical biochemistry JO - Anal Biochem VL - 258 IS - 1 N2 - 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. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/9527849/Measurement_of_Na+_K+_ATPase_activity_in_human_skeletal_muscle_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(98)92572-6 DB - PRIME DP - Unbound Medicine ER -