Further analysis of mutant thiolase protein in fibroblasts from a Japanese boy with 3-ketothiolase deficiency.
Further analysis of mutant thiolase protein in fibroblasts from a Japanese boy with 3-ketothiolase deficiency.
复制标题
进一步分析患有 3-酮硫解酶缺陷的日本男孩成纤维细胞中的突变硫解酶蛋白。
DOI：
10.1620/tjem.167.143
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
T. Hashimoto
中科院分区：
文献类型：
--
作者：
S. Yamaguchi;T. Fukao;M. Kano;A. Wakazono;T. Orii;N. Sakura;T. Hashimoto
我们检测了线粒体乙酰乙酰-辅酶A硫解酶突变蛋白(突变型T2)在一名患有3-酮硫解酶缺乏症的日本男孩的成纤维细胞中。经脉冲标记和SDS/PAGE测定，突变体T2蛋白的分子大小介于成熟亚基和T2前体之间。为了确定突变型T2蛋白的特性，我们进行了脉冲标记和罗丹明6G抑制成纤维细胞线粒体转运的实验，并通过硫解酶分析、免疫印迹和脉冲标记进行了家系研究。突变体T2早在10分钟的脉冲中就能被检测到。在罗丹明6G抑制实验和无细胞翻译实验中都没有检测到突变体T2的可能前体。在亲本中，乙酰乙酰-辅酶A硫解酶活性对K+离子的依赖性较低，免疫印迹的T2带较暗。因此，父母似乎是这种疾病的杂合子。在脉冲标记中，在患者中只检测到突变T2的一条带，在父亲中检测到正常的T2成熟亚单位的一条带；在母亲中检测到两条带。这些发现表明，患者的突变T2是从母亲那里遗传来的，父亲的另一个突变等位基因的表达可能被取消或很少。
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.