Chang D Y, Hou C, Bragg P D
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Arch Biochem Biophys. 1992 Mar;293(2):246-53. doi: 10.1016/0003-9861(92)90392-a.
Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.
将来自大肠杆菌JM83的外翻膜囊泡中吡啶核苷酸转氢酶的供能过程与用过量表达该酶的质粒pDC21转化的同一菌株的囊泡中的过程进行了比较。通过探针喹吖因荧光的猝灭来测定质子转运。能够消除跨膜质子梯度的试剂，如尼日利亚菌素以及解偶联剂3,3',4',5-四氯水杨酰苯胺和羰基氰化物间氯苯腙，抑制了NADH对NADP的ATP依赖性转氢作用，并消除了由NADPH对乙酰辅酶A的转氢作用、NADH的氧化以及ATP的水解所产生的跨膜质子梯度。在JM83和JM83pDC21这两种菌株的外翻膜囊泡中均观察到了这种情况。这两种菌株在依赖NADH氧化的转氢酶的反应方面存在显著差异。在JM83的膜囊泡中，该反应受到尼日利亚菌素和解偶联剂的抑制，但在JM83pDC21中几乎没有抑制作用，或者该反应受到刺激，尽管在后一种菌株中通过喹吖因荧光测定的由NADH氧化产生的质子梯度被消除了。有人提出，在该菌株的膜中，转氢酶是通过直接或局部（非体相）质子转运来供能的。解偶联剂可能会促进这些途径，但不会使其消除。使用其他菌株证明了这些观察结果的普遍性。与JM83相比，JM83pDC21的膜囊泡中的NADH氧化酶活性低几倍。两种菌株中泛醌和细胞色素的水平，以及NADH脱氢酶I和II以及细胞色素氧化酶的活性相似。得出的结论是，JM83pDC21的NADH氧化酶活性较低是因为该菌株膜中大量的转氢酶蛋白导致呼吸链电子传递复合物之间的碰撞速率降低。大量的转氢酶有利于直接的、非体相质子转移。转氢酶活性受到Ca2+、Mg2+或Mn2+的刺激。
