测定人体肌肉中完全活化的丙酮酸脱氢酶复合体的活性:评价一种有用的测定方法。

Enzyme & protein Pub Date : 1993-01-01 DOI:10.1159/000468654
W Sperl, J M Trijbels, W Ruitenbeek, H L van Laack, A J Janssen, C M Kerkhof, R C Sengers
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引用次数: 30

摘要

本文描述了一种灵敏的放射化学方法,用于测定骨骼肌组织中丙酮酸脱氢酶复合物(PDHC)的活性,该方法基于[1-14C]-丙酮酸脱羧为14CO2。测量既可以在肌肉匀浆中进行,也可以在600克上清中进行,两者都可以从小肌肉活检标本(20毫克)中获得。除培养液中的NAD+、焦磷酸硫胺素和辅酶A外,NADH:细胞色素c还原酶(NADHCR)与细胞色素c一起制备对PDHC活性有刺激作用。NADHCR构成NADH的氧化系统以防止反馈抑制。加入左旋肉碱也通过捕获生成的乙酰辅酶a作为乙酰左旋肉碱而导致PDHC的刺激。需要特别注意放射性丙酮酸盐,在-20℃的氮气条件下冷冻干燥和储存,并在每次PDHC检测期间测定纯度。在本实验中发现丙酮酸的Km值为0.084 mmol/l。非s型动力学,Hill系数为1.63。用所描述的方法,测量了完全Mg2+,Ca(2+)刺激的PDHC活性。添加纯化的特定丙酮酸脱氢酶磷酸酶并没有产生更高的PDHC活性。最后,比较总PDHC活性和[1-14C]-丙酮酸氧化速率,两者都是在新鲜肌肉制备的上清液中测量的,显示出等摩尔相关性,表明总PDHC活性在丙酮酸氧化速率的测定中是限速的。新生儿肌肉的PDHC活性和丙酮酸氧化率比对照组低5 - 10倍(年龄> 3岁)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Measurement of totally activated pyruvate dehydrogenase complex activity in human muscle: evaluation of a useful assay.

A sensitive radiochemical method for the determination of the pyruvate dehydrogenase complex (PDHC) activity in skeletal muscle tissue, based on the decarboxylation of [1-14C]-pyruvate to 14CO2, is described. Measurements can be carried out either in muscle homogenate or in 600-g supernatant, both obtainable from a small muscle biopsy specimen (20 mg). In addition to NAD+, thiamine pyrophosphate and coenzyme A in the incubation mixture, a preparation of NADH:cytochrome c reductase (NADHCR) together with cytochrome c has a stimulating effect on the PDHC activity. NADHCR constitutes an oxidation system for NADH to prevent feedback inhibition. Addition of L-carnitine also results in stimulation of PDHC by trapping the produced acetyl-CoA as acetylcarnitine. Special care for radioactive pyruvate, with freeze drying and storage at -20 degrees C under nitrogen, and determination of the purity during every PDHC assay, is required. In the presented assay a Km value of 0.084 mmol/l was found for pyruvate. Nonsigmoidal kinetics was found with a Hill coefficient of 1.63. With the described method, a totally Mg2+,Ca(2+)-stimulated PDHC activity is measured. Addition of a purified specific pyruvate dehydrogenase phosphatase did not yield a higher PDHC activity. Finally, comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that total PDHC activity is rate limiting in the assay for the pyruvate oxidation rate. Neonatal muscle exhibits five to ten times lower PDHC activities and pyruvate oxidation rates than controls (age > 3 years).

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