Robinson M S, Danson M J, Weitzman P D
Biochem J. 1983 Jul 1;213(1):53-9. doi: 10.1042/bj2130053.
Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.
首次从革兰氏阳性细菌（巨大芽孢杆菌）中纯化出了具有同质性的柠檬酸合酶。天然酶的相对分子质量测定为84000（标准误±5000）。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和在氯化胍中的凝胶过滤显示，存在一种相对分子质量为40300（标准误±4400）的单一蛋白质，表明该酶为二聚体。通过用辛二酸二甲酯和戊二醛使天然酶交联，随后进行电泳分析，证实了这种二聚体结构。该酶对两种底物乙酰辅酶A和草酰乙酸均遵循米氏动力学，并且对一系列腺嘌呤核苷酸的非特异性抑制敏感。在分子和催化特性方面，柠檬酸合酶与真核生物来源的酶非常相似，与革兰氏阴性细菌中较大的六聚体酶形成显著对比。
