Chretien D, Pourrier M, Bourgeron T, Séné M, Rötig A, Munnich A, Rustin P
Unité de Recherches sur les Handicaps Génétiques de l'Enfant, INSERM U-393, Hôpital des Enfants-Malades, Paris, France.
Clin Chim Acta. 1995 Sep 15;240(2):129-36. doi: 10.1016/0009-8981(95)06145-6.
In mitochondria-enriched preparations of human skeletal muscle, the measurement of pyruvate dehydrogenase activity, as determined by conventional spectrophotometric assay of NADH accumulation, is underestimated due to the oxidizing activity of the contaminating lactate dehydrogenase. Using a model reaction system consisting of varying mixtures of purified lactate and pyruvate dehydrogenases, we found that the presence of oxamate, a competitive inhibitor of the lactate dehydrogenase, allowed the measurement of a linear rate of pyruvate dehydrogenase activity without interference from lactate dehydrogenase. In the presence of 25 mM oxamate, this holds true up to a ratio of 30:1 for lactate to pyruvate dehydrogenases, respectively. A similar result was obtained when using human skeletal muscle mitochondria contaminated by lactate dehydrogenase. Rates of pyruvate dehydrogenase activity ranging from 50 to 120 nmol/min/mg protein could be routinely measured in such mitochondrial fractions. We concluded that the use of oxamate allows a spectrophotometric assay for pyruvate dehydrogenase activity to be utilized when screening for pyruvate dehydrogenase deficiency in mitochondria-enriched preparations of human skeletal muscle.
在富含线粒体的人骨骼肌制剂中，通过对NADH积累进行传统分光光度测定法来测定丙酮酸脱氢酶活性时，由于污染的乳酸脱氢酶的氧化活性，该测定值被低估。使用由纯化的乳酸脱氢酶和丙酮酸脱氢酶的不同混合物组成的模型反应系统，我们发现乳酸脱氢酶的竞争性抑制剂草氨酸盐的存在使得能够测量丙酮酸脱氢酶活性的线性速率，而不受乳酸脱氢酶的干扰。在存在25 mM草氨酸盐的情况下，对于乳酸脱氢酶与丙酮酸脱氢酶的比例分别高达30:1时，情况依然如此。当使用被乳酸脱氢酶污染的人骨骼肌线粒体时，也获得了类似的结果。在此类线粒体组分中，可以常规测量丙酮酸脱氢酶活性速率，范围为50至120 nmol/min/mg蛋白质。我们得出结论，在筛查富含线粒体的人骨骼肌制剂中的丙酮酸脱氢酶缺乏症时，使用草氨酸盐可使分光光度法用于丙酮酸脱氢酶活性的测定。
