Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme A mutase activity: HPLC and radioisotopic assays
Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme A mutase activity: HPLC and radioisotopic assays
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DOI：
10.1016/s0955-2863(98)00083-7
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发表时间：
1999-01-01
影响因子：
5.6
通讯作者：
Lambert, D
中科院分区：
文献类型：
--
作者：
Gaire, D;Sponne, I;Lambert, D
甲基丙二酰辅酶A（MCM）是一种5 '-脱氧腺苷钴胺素连接的线粒体酶，催化L-甲基丙二酰辅酶A异构化为琥珀酰辅酶A。总MCM活性和全MCM活性的体外测定是研究钴胺素途径的重要工具。已经描述了几种用于测量MCM活性的方法。最常用的方法是基于DL[CH 3-C-14]甲基丙二酰辅酶A的高锰酸盐氧化的放射性测定法，但放射性测定法不灵敏、费力且耗时。因此，我们比较了这种方法与nonradiometric测定，潜在的最敏感的，甲基丙二酰辅酶A和琥珀酰辅酶A的分离的基础上，通过高效液相色谱法（HPLC）。我们确定了最佳的测定条件和每种技术的重现性和灵敏度。这两种技术得到的结果非常不同：高锰酸盐氧化法比活度（全MCM活性为0.039 +/-0.013 nmol/min/mg蛋白质，总MCM活性为1.90 +/-0.69 nmol/min/mg蛋白质）比用HPLC方法获得的那些低三倍（全MCM活性为0.124 +/-0.011 nmol/min/mg蛋白质，总MCM活性为6.15 +/-0.76 nmol/min/mg蛋白质）。放射性测定法的变异系数（18.4-40.6%）是HPLC测定法的变异系数（3.5-12.2%）的3 - 5倍。这表明高锰酸盐放射性测定缺乏灵敏度和重现性。因此，放射性测量方法不适合于测量低放射性活度，例如组织中的空穴活度。放射性测定的固有不便表明，HPLC方法是测量MCM活性的首选方法。(J. Nutr. 10：56-62，1999）（C）Elsevier Science Inc. 1999. All rights reserved.
Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. In vitro assays of total and holo-MCM activities are important tools for investigating the cobalamin pathway. Several methods have been described for measuring MCM activity. The most commonly-used method is a radioassay based on the permanganate oxidation of DL[CH3-C-14]methylmalonyl-coenzyme A, but radiometric methods are insensitive, laborious and time-consuming. Therefore, we have compared this method with a nonradiametric assay, potentially most sensitive, based on the separation of methylmalonyl-coenzyme A and succinyl-coenzyme A by high performance liquid chromatography (HPLC). We determined the optimal assay conditions and the reproducibility and sensitivity of each technique. The results obtained by the two techniques were very different: the specific activities obtained by the permanganate oxidation method (0.039 +/- 0.013 nmol/min/mg protein for the holo-MCM activity and 1.90 +/- 0.69 nmol/min/mg protein for the total-MCM activity) were threefold lower than those obtained with the HPLC method (0.124 +/- 0.011 nmol/min/mg protein for the holo-MCM activity and 6.15 +/- 0.76 nmol/min/mg protein for the total-MCM activity). The coefficients of variation for the radiometric method (18.4-40.6%) were three to five times greater than those for the HPLC assay (3.5-12.2%). This demonstrates the lack of sensibility and reproducibility of the permanganate radioassay. Thus, the radiometric method is not suitable for measuring low mutase activities such as the hole activities in tissues. The intrinsic inconvenience of the radiometric assay indicates that the HPLC method is a method of choice for measuring MCM activity. (J. Nutr. Biochem. 10:56-62, 1999) (C) Elsevier Science Inc. 1999. All rights reserved.