Tayeh M A, Madigan M T
J Bacteriol. 1987 Sep;169(9):4196-202. doi: 10.1128/jb.169.9.4196-4202.1987.
The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in malate dehydrogenase specific activity, depending on the species, with recoveries ranging from 30 to 70%. Homogeneity of malate dehydrogenase preparations from the four organisms was determined by sodium dodecyl sulfate and nondenaturing polyacrylamide gel electrophoresis; a single protein band was observed in purified preparations by both techniques. The molecular weight of native malate dehydrogenases was determined by four independent methods and estimated to be in the range of 130,000 to 140,000 for the enzyme from R. capsulatus, R. rubrum, and R. vannielii and 57,000 for that from R. purpureus. It is concluded that malate dehydrogenase from R. capsulatus, R. rubrum, and R. vannielii is a tetramer composed of four identical subunits, while the enzyme from R. purpureus is a dimer composed of two identical subunits.
从非硫紫色细菌荚膜红细菌、深红红螺菌、万氏红微菌和紫红红环菌中纯化出了柠檬酸循环酶苹果酸脱氢酶，使其达到了均一性。通过单步或两步方案从每个物种中纯化了苹果酸脱氢酶：在所有情况下，三嗪染料亲和色谱法都是纯化苹果酸脱氢酶的关键步骤。纯化苹果酸脱氢酶后，其比活性提高了130至240倍，具体取决于物种，回收率在30%至70%之间。通过十二烷基硫酸钠和非变性聚丙烯酰胺凝胶电泳确定了来自这四种生物体的苹果酸脱氢酶制剂的均一性；两种技术在纯化制剂中均观察到一条单一的蛋白带。通过四种独立方法测定了天然苹果酸脱氢酶的分子量，估计荚膜红细菌、深红红螺菌和万氏红微菌的该酶分子量在130,000至140,000范围内，紫红红环菌的该酶分子量为57,000。得出的结论是，荚膜红细菌、深红红螺菌和万氏红微菌的苹果酸脱氢酶是由四个相同亚基组成的四聚体，而紫红红环菌的该酶是由两个相同亚基组成的二聚体。
