Molecular and biochemical study on multiple carboxylase deficiency.

多种羧化酶缺乏症的分子和生化研究。

• 批准号: 02454266
• 负责人: NARISAWA Kuniaki
• 金额: $ 4.35万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454266/
• 关键词: multiple carboxylase deficiency; biotin dependency; holocarboxylase synthetase; enzyme purification; enzyme diagnosis

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.

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。对胰蛋白酶肽进行高效液相色谱分离，并对其中四种分离的肽进行氨基酸分析。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。

项目成果

Chiba,Y.: "Purification and properties of holocarboxylase synthetase."

Chiba,Y.：“全羧化酶合成酶的纯化和特性。”

KURE,S.NARISAWA,K.TADA,K.: "ENZYMATIC DIAGNOSIS OF NONKETOTIC HYPERGLYCINEMIA;A NOVEL ASSAY OF GLYCINE CLEAVAGE SYSTEM ACTIVITY USING LYMPHOBLASTS TRANSFORMED BY EPSTEINーBARR VIRUS." J.PEDIATR.

KURE,S.NARISAWA,K.TADA,K.：“非酮症高甘氨酸血症的酶促诊断；利用 Epstein-Barr 病毒转化的淋巴细胞进行甘氨酸裂解系统活性的新颖测定。”

Suzuki, Y.: "Neonatal form of biotin-responsive multiple carboxylase deficiency." Journal of Nutritional Science and Vitaminology. 38. (1992)

Suzuki, Y.：“新生儿形式的生物素反应性多重羧化酶缺乏症。”

Chiba, Y.: "Purification and properties of holocarboxylse synthetase."

Chiba, Y.：“全羧基酶合成酶的纯化和特性。”

Suzuki, Y.: "Abnormal intake of vitamins : Biotin." Phama Medica. 10. (1992)

Suzuki, Y.：“维生素摄入异常：生物素。”