Molecular and biochemical study on multiple carboxylase deficiency.

新生儿多重羧化酶缺乏症在生命的最初几天表现为危及生命的酸中毒疾病。我们已经证明是由于全羧化酶合成酶（HCS）的缺陷，这是必不可少的附着生物素的非活性脱羧酶。发现来自患者的成纤维细胞具有PCC、MCC、PC和ACC的活性缺陷，并且具有异常的HCS活性，其对生物素的Km高度升高。用硫酸铵分级、Almina Cr分级、DEAE-SepharoseCL-6 B、EAH-Sepharose 4 B、Sephacryl S-200 HR、Hydroxyapatite HTP和Phenyl-Superose HR 5/5层析，从牛肝胞液中纯化出几乎均一的HCS。本研究采用了一种新的HCS检测方法，利用HCS缺陷患者培养的淋巴母细胞中的丙酰辅酶A脱羧酶作为底物。纯化后的酶在SDS PAGE上显示单一蛋白带，分子量为64，000。HCS是一种单计量蛋白。生物素的表观Km值为58 nM，ATP的表观Km值为28.6 μ M。HCS的胰蛋白酶消化，反相。进行胰蛋白酶肽的HPLC分离，以及分离的肽中的四种的氨基酸分析。用合成的寡核苷酸探针从牛肝cDNA文库中克隆了编码HCS的cDNA。

Neonatal multiple carboxylase deficiency presents as life-threatening acidotic illness in the earliest days of life. We have shown to be due to a defect in the enzyme holocarboxylase synthetase (HCS) which is essential for the attachment of biotin to the inactive apocarboxylase enzymes. Fibroblasts from a patient were found to have deficient activities of PCC, MCC, PC and ACC and have abnormal HCS activity with a highly elevated Km for biotin. HCS has been purified in nearly homogeneous form from bovine liver cytosol by the sequence of ammonium sulfate fractionation, Almina Cr fractionation, DEAE-SepharoseCL-6B, EAH-Sepharose 4B, Sephacryl S-200 HR, Hydroxyapatite HTP and Phenyl-Superose HR 5/5 chromatographies. A novel HCS assay method was adopted for this study utilizing propionly-CoA apocarboxylase from cultured lymphoblasts of HCS deficient patient as the substrate. The purified enzyme showed a single protein band on SDS PAGE with a molecular weight of 64, 000. HCS is a monometric protein. Its apparent Km values were 58 nM for biotin and 28.6 mu M for ATP. Tryptic digests of HCS, reverse-phase. HPLC separations of tryptic peptides, and amino acid analyses of four of the separated peptides were performed. A cDNA coding for the HCS was cloned from a bovine liver cDNA library by screening with synthetic oligonucleotide probes.