PURIFICATION AND PARTIAL CHARACTERIZATION OF THE GLYCINE DECARBOXYLASE MULTIENZYME COMPLEX FROM EUBACTERIUM-ACIDAMINOPHILUM
PURIFICATION AND PARTIAL CHARACTERIZATION OF THE GLYCINE DECARBOXYLASE MULTIENZYME COMPLEX FROM EUBACTERIUM-ACIDAMINOPHILUM
复制标题
DOI：
10.1128/jb.171.4.2209-2215.1989
复制
发表时间：
1989-04-01
影响因子：
3.2
通讯作者：
ANDREESEN, JR
中科院分区：
文献类型：
--
作者：
FREUDENBERG, W;ANDREESEN, JR
甘氨酸裂解系统的蛋白质 P1、P2 和 P4 已从厌氧、利用甘氨酸的细菌嗜酸氨基真杆菌中纯化。通过凝胶过滤，确定这些蛋白质的Mrs分别为225,000、15,500和49,000。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，确定蛋白质P1具有两个亚基，Mrs分别为59,500和54,100，表明α2.β2四聚体，而蛋白质P2和P4仅显示单条带，估计Mrs分别为15,500和42,000。在重构测定中，必须存在蛋白质 P1、P2、P4 和先前报道的硫辛酰胺脱氢酶 (P3)，才能实现甘氨酸脱羧酶或合酶活性。当在甘氨酸合酶反应中使用 NADP+ 作为电子受体或使用 NADPH 作为电子供体时，所有四种甘氨酸脱羧酶蛋白都表现出最高的活性。甘氨酸的氧化取决于四氢叶酸、二硫赤藓糖醇、NAD(P)+ 和磷酸吡哆醛的存在。后者与纯化的蛋白 P1 松散结合，仅与蛋白 P2 结合才能催化甘氨酸-碳酸氢盐交换反应。尽管硫辛酸被确定为蛋白质 P2 的组成部分（0.66 mol/mol 蛋白质），但蛋白质 P2 不能被硫辛酸或硫辛酰胺替代。四种分离蛋白和粗提物中的甘氨酸合酶活性较低，仅达到甘氨酸脱羧酶活性的 12%。针对 P1 和 P2 的抗体显示出与圆筒孢梭菌粗提物的交叉反应性。
The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an .alpha.2.beta.2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.