Brabson J S, Switzer R L
J Biol Chem. 1975 Nov 25;250(22):8664-9.
Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.
来自枯草芽孢杆菌的天冬氨酸转氨甲酰酶已被纯化至表观均一。通过在含有十二烷基硫酸钠的聚丙烯酰胺凝胶中电泳以及对溶解在6M盐酸胍中的蛋白质进行沉降平衡分析，得到亚基分子量为33,500±1,000。通过沉降速度和沉降平衡分析确定天然酶的分子量为102,000±2,000。因此，天冬氨酸转氨甲酰酶似乎是一种三聚体蛋白；用亚氨酯二甲酯交联并进行电泳分析证实了这种结构。枯草芽孢杆菌天冬氨酸转氨甲酰酶的氨基酸组成与大肠杆菌天冬氨酸转氨甲酰酶催化亚基的氨基酸组成非常相似；只有四种氨基酸的含量有显著差异。变性酶有一个游离巯基。天冬氨酸转氨甲酰酶表现出米氏动力学，并且不受核苷酸抑制或激活。几种阴离子可将活性提高2至5倍。免疫化学研究表明，枯草芽孢杆菌与大肠杆菌天冬氨酸转氨甲酰酶或大肠杆菌天冬氨酸转氨甲酰酶催化亚基之间几乎没有相似性。
