Laubinger W, Dimroth P
Institut für Physiologische Chemie der Technischen Universität München, Federal Republic of Germany.
Biochemistry. 1988 Sep 20;27(19):7531-7. doi: 10.1021/bi00419a053.
The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.
中度嗜丙酸菌的ATP合酶（F1F0）已被纯化至比细菌膜高约6倍的5.5单位/毫克蛋白质的比ATP酶活性。SDS凝胶电泳分析表明，除了F1 ATP酶的五个亚基外，还纯化了分子量为26,000（a）、23,000（b）和7500（c）的亚基。F1F0的ATP酶活性被Na+离子特异性激活约10倍。该酶受到二环己基碳二亚胺、venturicidin、三丁基氯化锡和叠氮化物的强烈抑制。用[14C]二环己基碳二亚胺孵育后，每500,000克酶结合约3 - 4摩尔抑制剂。放射性标记特异性结合到亚基c上。这些亚基形成稳定的聚集体，在100℃下能抵抗SDS的解离。单体是在与SDS一起加热至121℃或用氯仿/甲醇提取膜后形成的。通过冻融超声处理将ATP合酶整合到脂质体中。重组的蛋白脂质体在ATP水解时催化Na+离子的转运。二环己基碳二亚胺完全消除了这种转运。莫能菌素阻止了Na+离子的积累，而在缬氨霉素或羰基氰化物m -氯苯腙存在下，摄取速率提高了4 - 5倍。这些结果表明存在电致Na+转运，并且这是一个初级事件，不是由H+转运ATP合酶与Na+/H+反向转运体共同完成的。
