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

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 reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

Capillary electrophoresis-based assay of phosphofructokinase-1

“…This leaves the difference in Mg 2+ concentration between the sample solution (5 mM) and separation buffer (1 mM) as the most likely cause. An alternative approach to reduce the negative impact of metal binding to ADP and ADP on the separation is to add EDTA to the separation buffer [30; 31], and this approach has been used in the development of a CE enzyme assay [32]. …”

Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors

“…Capillary electrophoresis methods were optimized and validated for the determination of phosphorylated TK products and subsequently used for setting up a fast and easy CE-based TK assay. Several methods have previously been developed for the separation and quantitation of nucleosides and nucleotides after enzymatic reactions [26][27][28][29][30][31]. Theses studies were taken as a starting point for adaptation to the biological project of monitoring HSV-1 TK.…”

Capillary Electrophoresis as a Monitoring Tool for Flow Composition Determination

“…The same FIA–CE interface with membrane valves and gas exchange pumps (controlled by solenoid valves) has been used for studying the flows originating from various bioprocesses. Among these processes are monitoring of the enzymatic conversion of adenosine triphosphate to adenosine diphosphate [ 8 ], monitoring of the kinetics of the metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers [ 9 ], monitoring of the degradation of phenols by Rhodococcus bacteria [ 10 ], and monitoring of the bioaccumulation of Cu, Zn, Co, and Cd by the Rhodococcus sp. bacteria (isolated from highly polluted technogenic soil in Estonia) [ 11 ].…”