By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.
摘要
通过使用Bio-Gel A1.5M和Sephadex G - 150柱,对大肠杆菌的无细胞粗提取物进行分级分离,以证明脯氨酸生物合成聚集体的存在。2. Sephadex G - 150可分离出两种受脯氨酸抑制的谷氨酰胺激酶,分子量分别为125000和38000,其反应依赖于Mg2+。分子量较大的那种对脯氨酸抑制更敏感。3. γ-谷氨酰磷酸还原酶和1-吡咯啉-5-羧酸还原酶(EC 1.5.1.2)的分子量分别约为125000和190000,后者的比活性比其他两种生物合成酶或体内整个途径的比活性高5×10³倍。4. Bio-Gel A1.5M色谱法得到一种分子量为250000的单一谷氨酰胺激酶,并讨论了其作为酶复合物成分的可能性。