Verity M A, Turnbull D M
Department of Pathology (Neuropathology), UCLA Center for Health Sciences 90024-1732.
Biochem Med Metab Biol. 1993 Jun;49(3):351-62. doi: 10.1006/bmmb.1993.1036.
The acyl-CoA dehydrogenases (ACDs) are mitochondrial enzymes that dehydrogenate acyl-coenzyme A esters of different chain lengths. Inherited deficiencies of these dehydrogenases are commonly associated with muscle weakness and lipid storage. Numerous assays including spectrophotometric, fluorometric, chemical, and radiochemical procedures have been used, but there is need for a rapid, reproducible assay for the different acyl-CoA dehydrogenases in small frozen samples of human muscle biopsies. We describe a comparative study of dye-linked spectrophotometric assays of the long, medium, and short chain acyl-CoA dehydrogenases in frozen rat and human muscle samples. An optimal procedure is described confirming the value of glass-glass homogenization and assay of a 600g supernatant. Higher activities for all acyl-CoA dehydrogenases, citrate synthase, and cytochrome c oxidase were obtained in rat in contrast to human. The substrate-linked dye reduction method was found superior to the ferricenium or electron transfer flavoprotein acceptor systems. Application of the phenazine ethosulfate-DCPIP-linked method to medium-chain acyl-CoA dehydrogenase (MCAD) was studied in detail and the effect of immunoprecipitation of MCAD allowed for the determination of substrate specificity and the degree of crossover between long-, medium-, and short-chain ACD activity following immunoprecipitation. Finally, a comparison of the specificity and validity of the assay in a patient with MCAD deficiency was performed.
酰基辅酶A脱氢酶（ACD）是一种线粒体酶，可使不同链长的酰基辅酶A酯脱氢。这些脱氢酶的遗传性缺陷通常与肌肉无力和脂质储存有关。已经使用了多种检测方法，包括分光光度法、荧光法、化学法和放射化学法，但需要一种快速、可重复的检测方法来检测人肌肉活检小冷冻样本中的不同酰基辅酶A脱氢酶。我们描述了一项对冷冻大鼠和人肌肉样本中长链、中链和短链酰基辅酶A脱氢酶的染料偶联分光光度法检测的比较研究。描述了一种优化的方法，证实了玻璃-玻璃匀浆和检测600g上清液的价值。与人类相比，大鼠中所有酰基辅酶A脱氢酶、柠檬酸合酶和细胞色素c氧化酶的活性更高。发现底物偶联染料还原法优于铁茂或电子传递黄素蛋白受体系统。详细研究了吩嗪硫酸乙酯-DCPIP偶联法在中链酰基辅酶A脱氢酶（MCAD）检测中的应用，MCAD免疫沉淀的效果有助于确定底物特异性以及免疫沉淀后长链、中链和短链ACD活性之间的交叉程度。最后，对一名MCAD缺乏症患者检测方法的特异性和有效性进行了比较。
