Molecular and biochemical study on multiple carboxylase deficiency.

新生儿多重羧化酶缺乏症在生命早期表现为危及生命的酸中毒疾病。我们已经证明，这是由于一个缺陷的酶全息羧化酶合成酶（HCS），这是必不可少的附着的生物素到无活性的羧化酶。1例患者的成纤维细胞发现PCC、MCC、PC和ACC活性不足，HCS活性异常，生物素Km值高。通过硫酸铵分馏、Almina Cr分馏、DEAE-SepharoseCL-6B、EAH-Sepharose 4B、Sephacryl S-200 HR、羟基磷灰石HTP和Phenyl-Superose HR 5/5层析，从牛肝细胞质中纯化出了HCS。本研究采用一种新的HCS检测方法，以培养的HCS缺陷患者淋巴细胞丙酸辅酶A羧化酶为底物。纯化后的酶在SDS PAGE上显示一条蛋白带，分子量为64000。HCS是一种单量蛋白。生物素表观Km值为58 nM， ATP表观Km值为28.6 μ M。HCS的胰蛋白酶消化，反相。高效液相色谱法分离色氨酸肽，并对分离的四种肽进行氨基酸分析。通过人工合成的寡核苷酸探针筛选，从牛肝脏cDNA文库中克隆出一个编码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.