Ito J, Cox E C, Yanofsky C
J Bacteriol. 1969 Feb;97(2):725-33. doi: 10.1128/jb.97.2.725-733.1969.
A procedure employed in the purification of anthranilate synthetase component I of Escherichia coli is described. The purified component appears homogeneous by starch gel electrophoresis and by sedimentation analysis. A molecular weight of 60,000 was estimated by gel filtration of Sephadex G-100. This value is consistent with the molecular weight estimated from the sedimentation and diffusion coefficients. Purified anthranilate synthetase component I cannot use glutamine as substrate and thus has no activity in the reaction of chorismate + l-glutamine --> anthranilate; however, it is active when ammonium sulfate is provided as amino donor. Sucrose density gradient analyses showed that ammonium sulfate does not affect the sedimentation velocity of component I. The ultraviolet absorption and fluorescence spectra of the purified component indicated that it contains tryptophan. Peptide pattern and extract complementation evidence suggested that the protein is a single polypeptide chain. Enzyme activity measurements indicated that wild-type E. coli produces equimolar amounts of at least four of the five polypeptides specified by the operon. Purified anthranilate synthetase component I is inhibited by l-tryptophan.
本文描述了一种用于纯化大肠杆菌邻氨基苯甲酸合成酶组分I的方法。通过淀粉凝胶电泳和沉降分析，纯化后的组分看起来是均一的。通过Sephadex G - 100凝胶过滤法估计其分子量为60,000。该值与根据沉降系数和扩散系数估计的分子量一致。纯化后的邻氨基苯甲酸合成酶组分I不能使用谷氨酰胺作为底物，因此在分支酸 + L - 谷氨酰胺→邻氨基苯甲酸的反应中没有活性；然而，当提供硫酸铵作为氨基供体时，它具有活性。蔗糖密度梯度分析表明，硫酸铵不影响组分I的沉降速度。纯化组分的紫外吸收光谱和荧光光谱表明它含有色氨酸。肽谱和提取物互补证据表明该蛋白质是一条单多肽链。酶活性测量表明，野生型大肠杆菌产生的由操纵子指定的五种多肽中至少有四种是等摩尔量的。纯化后的邻氨基苯甲酸合成酶组分I受到L - 色氨酸的抑制。
