Whelan A J, Strauss A W, Hale D E, Mendelsohn N J, Kelly D P
Department of Medicine, St. Louis Children's Hospital, Missouri 63110.
Pediatr Res. 1993 Nov;34(5):694-7. doi: 10.1203/00006450-199311000-00025.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a frequent and sometimes fatal inherited metabolic disorder of fatty acid beta-oxidation. A eukaryotic expression system was used to study naturally occurring mutations in MCAD. The 1263 nucleotide coding region of human MCAD cDNA was inserted downstream of the SV40 early promoter for high-level expression in Chinese hamster ovary cells. Both normal MCAD cDNA and a mutant MCAD cDNA containing the common, disease-causing A to G transition at position 985 (A985G), which alters a lysine to a glutamic acid (K304E), were inserted into expression vectors. Transient transfection of Chinese hamster ovary cells was performed with the expression constructs. The steady state level of expressed normal MCAD protein antigen was substantially higher (5-fold) than the expressed mutant protein. The MCAD enzymatic activity in protein extracts from cells containing the expressed normal MCAD cDNA was also much higher (6-fold) than the activity in cells expressing the mutant MCAD. Therefore, these data confirm that the common K304E mutation causes MCAD deficiency primarily by decreased protein stability rather than reduction of catalytic activity and, in fact, demonstrate that the K304E mutant protein has a similar sp act against octanoyl CoA substrate as the normal protein.
中链酰基辅酶A脱氢酶（MCAD）缺乏症是一种常见的、有时甚至会致命的脂肪酸β氧化遗传性代谢紊乱疾病。利用真核表达系统研究MCAD中的自然发生突变。将人MCAD cDNA的1263个核苷酸编码区插入SV40早期启动子下游，以便在中国仓鼠卵巢细胞中进行高水平表达。将正常MCAD cDNA和一个包含常见致病A到G转换（第985位，A985G）的突变MCAD cDNA插入表达载体，该转换将赖氨酸变为谷氨酸（K304E）。用表达构建体对中国仓鼠卵巢细胞进行瞬时转染。表达的正常MCAD蛋白抗原的稳态水平显著高于（5倍）表达的突变蛋白。含有表达的正常MCAD cDNA的细胞蛋白提取物中的MCAD酶活性也比表达突变MCAD的细胞中的活性高得多（6倍）。因此，这些数据证实，常见的K304E突变主要通过降低蛋白质稳定性而非降低催化活性导致MCAD缺乏，事实上，表明K304E突变蛋白对辛酰辅酶A底物的比活性与正常蛋白相似。
