Yamaguchi S, Fukao T, Kano M, Wakazono A, Orii T, Sakura N, Hashimoto T
Department of Pediatrics, Gifu University School of Medicine, Japan.
Tohoku J Exp Med. 1992 Jun;167(2):143-53. doi: 10.1620/tjem.167.143.
We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulse-labeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty.
我们研究了一名患有3-酮硫解酶缺乏症的日本男孩成纤维细胞中线粒体乙酰乙酰辅酶A硫解酶的突变蛋白（突变体T2）。通过脉冲标记和SDS/聚丙烯酰胺凝胶电泳测定，突变体T2蛋白的分子大小介于T2成熟亚基和前体之间。为了表征突变体T2蛋白，我们进行了成纤维细胞中线粒体运输的脉冲标记和罗丹明6G抑制实验、无细胞翻译实验，以及通过硫解酶测定、免疫印迹和脉冲标记进行的家系研究。早在10分钟的脉冲标记时就能检测到突变体T2。在罗丹明6G抑制实验或无细胞翻译实验中均未检测到突变体T2可能的前体。在父母中，乙酰乙酰辅酶A硫解酶活性对钾离子的依赖性较低，免疫印迹中的T2条带较淡。因此，父母似乎是这种疾病的杂合子。在脉冲标记实验中，在患者中仅检测到突变体T2的条带，在父亲中仅检测到T2正常成熟亚基的单一条带；在母亲中检测到了这两条带。这些发现表明，患者中的突变体T2是从母亲遗传而来的，并且父亲另一个突变等位基因的表达可能被消除或很少。
