In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

Electrophoresis
Maria KulpValdur Saks

Abstract

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and...Continue Reading

Citations

Feb 11, 2005·Analytical and Bioanalytical Chemistry·Melissa Gayton-ElyLisa A Holland
Feb 4, 2012·Analytical and Bioanalytical Chemistry·Tomáš Křížek, Anna Kubíčková
Jan 22, 2014·Analytical Biochemistry·Andrew MalinaS Douglass Gilman
Oct 7, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Jamshed IqbalChrista E Müller
Apr 1, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Zdenek Glatz
Nov 29, 2005·Journal of Chromatography. a·M KulpM Kaljurand
Aug 28, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Mihkel KaljurandJekaterina Mazina-Šinkar

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