Voprosy meditsinskoi khimii (ISSN 0042-8809)

Isolation, identification and quantitative determination of the ATP synthesized by a preparation of plasma membrane-enriched particles from rat skeletal muscles in the presence of insulin

   
Karelin A.A.
PubMed Id: 7032065
Year: 1981 vol: 27  issue:5  pages: 679-685
Abstract: ATP was synthesized in presence of insulin or insulin and prostaglandin E2 by rat skeletal muscle particulate preparation enriched in plasmatic membranes. Isolation of the ATP was carried out using column chromatography on Dowex 1 x 8/cl-form, 100--200 mesh). ATP was formed within 1 min in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, inorganic phosphate, NaF during NADH-related oxidation involving cytochrome c and O2 in amounts of 100--300 pmoles per mg of protein. Quantitative estimation of ATP in the lyophilized product was carried out by means of spectrophotometry at 340 nm of NADPH formed during a coupled enzymatic reactions involving hexokinase and glycose-6-phosphate dehydrogenase. This products identified by descending paper chromatography on Whatman NI in the system containing ethanol-ammonium citrate pH 4.4 and pH 7.5. Identification of ATP was also performed by thin-layer chromatography. The product was tested for content of ribose (orcinol method) and of inorganic phosphate after acid hydrolysis within 7.5 min at 100 degrees. In the product obtained adenine was identified by UV-spectrophotometry at 260 nm. A salt of ATP was synthesized from the product obtained.
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Reference: Karelin A.A., Isolation, identification and quantitative determination of the ATP synthesized by a preparation of plasma membrane-enriched particles from rat skeletal muscles in the presence of insulin, Voprosy meditsinskoi khimii, 1981, vol: 27(5), 679-685.
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