Immunotitration Analysis of Cytosolic Acetoacetyl-Coenzyme A Thiolase Activity in Human Fibroblasts
Immunotitration Analysis of Cytosolic Acetoacetyl-Coenzyme A Thiolase Activity in Human Fibroblasts
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人成纤维细胞胞质乙酰乙酰辅酶 A 硫解酶活性的免疫滴定分析
DOI：
10.1203/00006450-199606000-00020
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发表时间：
1996
影响因子：
3.6
通讯作者：
N. Kondo
中科院分区：
文献类型：
--
作者：
T. Fukao;Xiang;S. Yamaguchi;T. Hashimoto;T. Orii;N. Kondo
在人成纤维细胞中有五种已知的硫解酶，除了线粒体三功能蛋白（TFP）外，所有的硫解酶都对乙酰乙酰辅酶A（AACoA）具有硫解酶活性。我们研究了线粒体乙酰乙酰辅酶A硫解酶（AACoAT）（T2）、胞质AACoAT（CT）和线粒体3-酮脂酰辅酶A硫解酶（T1）对对照人成纤维细胞中总AACoAT活性的贡献。在对照成纤维细胞中用抗T2、CT或T1的抗体进行AACoAT活性的免疫滴定，结果如下。在不存在钾离子的AACoAT活性的情况下，总活性的26- 38%、40- 47%和11-20%分别来自CT、T1和T2。当联合使用三种抗体时，未对剩余的6-9%总活性进行免疫滴定。因此，在不存在钾离子的情况下，过氧化物酶体3-酮脂酰-CoA硫解酶对总AACoAT活性的贡献至少小于6- 9%。由于总AACoAT活性的正常范围相对较宽，因此难以根据总AACoAT活性的降低来评估CT缺陷。在6个对照成纤维细胞中用抗CT抗体进行的免疫滴定显示CT活性范围在1.3和2.4 nmol/min/mg蛋白质之间。免疫滴定法被证明是一种准确的方法来评估CT活性。来自CT缺乏症患者的两种细胞系已经灭绝。
There are five known thiolases in human fibroblasts, and all but mitochondrial trifunctional protein (TFP) have thiolase activity toward acetoacetyl-CoA (AACoA). We investigated the contribution of mitochondrial acetoacetyl-CoA thiolase (AACoAT) (T2), cytosolic AACoAT (CT), and mitochondrial 3-ketoacyl-CoA thiolase (T1) to the total AACoAT activity in control human fibroblasts. Immunotitration of AACoAT activity with antibodies against T2, CT, or T1 was carried out in control fibroblasts, with the following results. In the case of AACoAT activity in the absence of potassium ion, 26-38%, 40-47%, and 11-20% of the total activity derived from CT, T1, and T2, respectively. The residual 6-9% total activity was not immunotitrated when three antibodies were used in combination. Hence, the contribution of peroxisomal 3-ketoacyl-CoA thiolase to the total AACoAT activity in the absence of potassium ion was at least less than 6-9%. Because the normal range of total AACoAT activity is relatively wide, it is difficult to evaluate CT defects based on a decrease of total AACoAT activity. Immunotitration with anti-CT antibody in six control fibroblasts revealed that CT activity ranges between 1.3 and 2.4 nmol/min/mg of protein. Immunotitration proved to be an accurate method to evaluate CT activity. The two cell lines from patients with CT deficiency have become extinct.
DOI：
10.1016/s0021-9258(18)48391-3
发表时间：
1992-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Yasushi Uchida;K. Izai;T. Orii;T. Hashimoto
通讯作者：
Yasushi Uchida;K. Izai;T. Orii;T. Hashimoto
影响因子：
15.9
作者：
KAMIJO, T;WANDERS, RJA;HASHIMOTO, T
通讯作者：
HASHIMOTO, T