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。我们已经证明这是由于全羧化酶合成酶 (HCS) 的缺陷造成的，HCS 对于生物素与无活性的脱辅基羧化酶的附着至关重要。发现患者的成纤维细胞 PCC、MCC、PC 和 ACC 活性不足，并且 HCS 活性异常，生物素 Km 高度升高。 HCS 已通过硫酸铵分级分离、Almina Cr 分级分离、DEAE-SepharoseCL-6B、EAH-Sepharose 4B、Sephacryl S-200 HR、羟基磷灰石 HTP 和 Phenyl-Superose HR 5/ 序列从牛肝细胞质中纯化为几乎均质的形式5 色谱图。本研究采用了一种新的 HCS 测定方法，以 HCS 缺陷患者培养的淋巴母细胞中的丙酰辅酶 A 脱辅基羧化酶为底物。纯化的酶在 SDS PAGE 上显示出单一蛋白条带，分子量为 64, 000。HCS 是一种单体蛋白。生物素的表观 Km 值为 58 nM，ATP 的表观 Km 值为 28.6 muM。 HCS 的胰蛋白酶消化物，反相。对胰蛋白酶肽进行 HPLC 分离，并对四种分离的肽进行氨基酸分析。通过用合成寡核苷酸探针筛选，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。”

KURE,S.: "STRUCTURAL AND EXPRESSION ANALYSIS OF NORMAL AND MUTANT mRNA ENCODING GLYCINE DECARBOXYLASE:THREE BASE DELETION IN mRNA CAUSES NONKETOTIC HYPERGLYCINEMIA." BIOCHEM.BIOPHYS.RES.COMMUN.

KURE,S.：“编码甘氨酸脱羧酶的正常和突变 mRNA 的结构和表达分析：mRNA 中的三碱基缺失导致非酮性高甘氨酸血症。”