Barash H, Halpern Y S
Biochim Biophys Acta. 1975 Mar 28;386(1):168-80. doi: 10.1016/0005-2795(75)90257-3.
Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).
通过溶菌酶 - EDTA处理从大肠杆菌K - 12周质空间释放的谷氨酸结合蛋白被纯化至同质，并对其物理和化学性质进行了研究。它是一种碱性蛋白，pI为9.1。通过分析超速离心、在Sephadex G - 100上的凝胶过滤以及十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定的分子量分别为29700、27800和32000。谷氨酸的KD值为6.7×10⁻⁶ M。L - 天冬氨酸、还原型谷胱甘肽、γ - 苄基 - L - 谷氨酸酯和γ - 乙基 - L - 谷氨酸酯竞争性抑制谷氨酸结合，其Ki值分别为7.8×10⁻⁵、1.1×10⁻⁵、1.0×10⁻⁵和1.0×10⁻⁵ M。与完整细胞相比，原生质球保留了40%的谷氨酸转运能力。利用谷氨酸的菌株（CS7）的谷氨酸结合活性是不利用谷氨酸的亲本菌株（CS101）的1.6倍。同样，温度条件性利用谷氨酸的突变体（CS2 - TC）在允许温度（42℃）下生长时的谷氨酸结合活性比在限制温度（30℃）下生长时高1.9倍。
