ACC检测方法.docVIP

Acetyl-CoA Carboxylase from Rat Liver EC 6.4.1.2 Acetyl-CoA: carbon-dioxide ligase (ADP-forming) By TADASHI TANABE, SHIGETADA NAKANISHI, TAKASHI HASHIMOTO, HIDEO OGIWARA, JUN-ICHI NIKAWA, and SHOSAKU NUMA ATP+HCO3-+acetyl-CoA ADP+Pi+malonyl-CoA Assay Methods The principles underlying the various assays of acetyl-CoA carboxylase have been described in previous articles in this series.1-4 Most conveniently, the enzyme activity is determined by 14CO2—fixation assay or by the spectrophotometric assay in combination with the pyruvate kinase and lactate dehydrogenase reactions. The 14CO2—fixation assay can be used for enzyme preparations from all steps, whereas the spectrophotometric assay is applicable to preparation from the DEAE-cellulose chromatography step and subsequent steps. 14CO2—Fixation Method Reagents Tris-HCl buffer, 0.5M, PH 7.5 Potassium citrate, 0.1M MgCl2, 0.1M Reduced glutathione, 0.1M, PH 7.5 Bovine serum albumin, 3% ATP, 0.5M Acetyl-CoA, 10mM KH14CO3 (0.25μCi/μmol), 0.2M HCl, 5M Scintillator solution: 4g of 2,5-diphenyloxazole and 0.1g of 1,4-bis [2-(4-methyl-5-phenyloxazolyl)]benzene in 1 liter of toluene plus o.5 liter of Triton X-100 Procedure: When the crude extract is assayed, it is passed through a Sephadex G-5 column to remove endogenous substrates. Because rat liver acetyl-CoA carboxylase requires preincubation with citrate to attain its full activation,5 the enzyme is first preincubated at 37℃ for 30 min in a mixture containing 50mM Tris-HCl buffer, PH 7.5, 10mM potassium citrate, 10mM MgCl2, 3.75mM glutathione, and 0.75mg of bovine serum albumin per milliliter. The reaction is then initiated by adding an aliquot of the preincubated enzyme (up to 0.2mU) to an assay mixture (final volume, 0.8ml) containing 50mM Tris-HCl buffer, PH 7.5, 10mM postassium citrate, 10mM MgCl2, 3.75mM ATP, 0.125mM acetyl-CoA, and 12.5mM KH14CO3(0.25μCi/μmol). After incubation at 37℃ for 10 min, the reaction is terminated with 0.2ml of 5M HCl. The reaction mi