Some properties of PMS-Lactate and NAD-Alcohol Dehydrogenases Participating in Supply of Electron to Bacterial Photosystem

紫色非硫细菌的有机物电子获得系统的研究资料有限。在沼泽红球藻7号的无细胞提取物中检测到NAD依赖性醇脱氢酶（ADH）和NAD非依赖性（PMS依赖性）D-和L-乳酸脱氢酶（D-LDH和L-LDH）。从这些酶中，D-LDH被纯化为电泳均一的蛋白质。经凝胶过滤层析和SDS-聚丙烯酰胺凝胶电泳测得D-LDH及其亚基的分子量分别约为235 kDa和57 kDa。pI为5.0。该酶的最适pH为8.5，最适温度为50℃。该酶对D-乳酸的Km为0.8mM，底物专一性较窄，对L-乳酸呈惰性。该酶被可逆地抑制草酸盐（Ki，0.12 mM）。酶的辅基为FAD。此外，从7号菌株的无细胞提取物中纯化的细胞色素是一种结构均一的蛋白质。其分子量估计约为12.4 kDa。经鉴定为细胞色素c_2（Cyt c_2）。D-LDH氧化D-乳酸时，细胞色素c_2（Cyt c_2）也被还原，说明乳酸的电子被利用，流向D-乳酸→D-LDH（FAD）→Cyt c_2→光系统→NAD，L-DH和ADH不稳定。这些酶尚未被纯化为均质蛋白质。

Information of electron gain system from organic compounds in purple nonsulfur bacteria is limited. NAD- dependent alcohol dehydrogenase (ADH), and NAD- independent (PMS-dependent) D-and L-lactate dehydrogenases (D-LDH and L-LDH) were detected in the cell-free extracts of Rhodopseudomonas palustris No.7. From these enzymes, D-LDH was purified as an electrophoretically homogeneous protein. The molecular weight of D-LDH and its subunit were estimated to be approximately 235 kDa and 57 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The pI was 5.0. The optimum pH for the enzyme activity was 8.5 and the optimum temperature was 50℃. The Km against D-lactate was 0.8 mM.The substrate specificity of the enzyme was narrow, and it was inert to L-lactate. The enzyme was reversibly inhibited by oxalate (Ki, 0.12 mM). The prosthetic group of the enzyme was clarified to be FAD.Furthermore, cytochrome in the cell-free extracts of strain No.7 was purified as an electrophoretically homogeneous protein. Its molecular weight was estimated to be approximately 12.4 kDa. It was identified as cytochrome c_2 (Cyt c_2). The reduction of Cyt c_2 was observed with the oxidation of D-lactate by D-LDH.From this, it was suggested that the electron from lactate was utilized in flowing to D-lactate→D-LDH (FAD)→Cyt c_2→photosystem→NAD.L-DH and ADH were unstable. These enzymes have not been purified as a homogeneous protein.