利用小鼠肝脏胞质溶胶组分和从马红细胞中纯化的可溶性细胞色素b5重建CMP-N-乙酰神经氨酸羟化活性。

Reconstitution of CMP-N-acetylneuraminic acid hydroxylation activity using a mouse liver cytosol fraction and soluble cytochrome b5 purified from horse erythrocytes.

作者信息

Kozutsumi Y, Kawano T, Kawasaki H, Suzuki K, Yamakawa T, Suzuki A

机构信息

Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science.

出版信息

J Biochem. 1991 Sep;110(3):429-35. doi: 10.1093/oxfordjournals.jbchem.a123598.

DOI:10.1093/oxfordjournals.jbchem.a123598

PMID:1769971

Abstract

The hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively.

摘要

在CMP-N-羟乙酰神经氨酸形成过程中，CMP-N-乙酰神经氨酸（CMP-NeuAc）的羟基化需要几个组成电子传递系统的成分。从马红细胞裂解物中纯化出一种替代其中一个成分的蛋白质，使其达到均质状态。基于其部分氨基酸序列和免疫交叉反应性，该蛋白质被鉴定为缺乏微粒体细胞色素b5膜结构域的可溶性细胞色素b5。重构了参与CMP-NeuAc羟基化的电子传递系统，然后使用纯化的马可溶性细胞色素b5和小鼠肝脏胞质溶胶中的一个组分对其进行表征。羟基化反应需要一种还原剂，二硫苏糖醇（DTT）最为有效。NADH或NADPH均可作为电子供体，但以NADPH时的活性约为以NADH时的74%。盐类和叠氮化物分别因中断从NAD(P)H到细胞色素b5的电子传递以及终端酶反应而抑制羟基化。