Dimroth P, Thomer A
Biol Chem Hoppe Seyler. 1986 Aug;367(8):813-23. doi: 10.1515/bchm3.1986.367.2.813.
Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.
肺炎克雷伯菌细胞在柠檬酸盐上厌氧生长后形成的菌悬液，其柠檬酸盐降解特别需要钠离子。柠檬酸盐降解速率远低于柠檬酸盐发酵酶柠檬酸盐裂解酶和草酰乙酸脱羧酶的活性，这表明柠檬酸盐转运是限速步骤。柠檬酸盐摄入细胞也依赖钠离子，且伴随着其快速代谢，因此三羧酸不会在细胞中大量积累。LiCl对转运的刺激效率较低。锂离子与柠檬酸盐共转运进入细胞。解偶联剂[4-(三氟甲氧基)苯腙]丙二腈(CCFP)可消除柠檬酸盐的转运和降解。通过冷渗透休克处理释放外膜成分和周质结合蛋白后，柠檬酸盐降解对莫能菌素和缬氨霉素也变得敏感。休克处理对柠檬酸盐降解速率没有影响，这表明转运不依赖于结合蛋白。在以柠檬酸盐为有氧生长底物的肺炎克雷伯菌以及以柠檬酸盐加葡萄糖为厌氧生长底物的大肠杆菌中柠檬酸盐降解和转运不依赖钠离子。通过转化编码柠檬酸盐转运的肺炎克雷伯菌基因获得的大肠杆菌cit+克隆，在以柠檬酸盐为底物进行有氧生长时特别需要钠离子，这表明依赖钠离子的柠檬酸盐转运系统在起作用。钠离子和锂离子在刺激大肠杆菌cit+细胞悬液的柠檬酸盐降解方面效果相同。大肠杆菌cit+膜囊泡中的柠檬酸盐转运也依赖钠离子，并由质子动力(δμH+)提供能量。离子载体使δμH+或其组分δpH或δψ消散，要么完全消除要么极大地抑制柠檬酸盐摄取。有人提出，在厌氧条件下为柠檬酸盐转运提供能量的系统，是由代谢终产物与质子的外向共转运产生δpH，以及由草酰乙酸脱羧产生δpNa+和δψ。在发酵柠檬酸盐的肺炎克雷伯菌中未发现由钠离子激活的ATP酶。然而，细胞在其膜中含有质子转运ATP酶和Na+/H+反向转运蛋白。
