We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na+-translocating (NQR) and H+-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na+ and Li+ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na+. These findings testified a primary Na+-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H+ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H+-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na+ or H+ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na+ transport in nitrogen fixation by A. vinelandii.

中文翻译：

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一种区分H +和Na +转运的NADH：醌氧化还原酶的简单策略。

我们在这里描述一种简单的策略来表征NADH：醌氧化还原酶的运输特异性，使用土壤细菌Azotobactervinelandii的Na +-易位（NQR）和H +-易位（NDH-1）酶作为模型。毫摩尔浓度的Na +和Li +会增加由NDH-1缺陷菌株制备的倒膜囊泡对deaminoNADH的氧化速率。在Na +存在下，囊泡产生了耐羰基氰化物间氯苯基（CCCP）的电势差和CCCP刺激的pH差（内部碱化）。这些发现证明了A.vinelandii NQR的主要Na +泵功能。此外，用荧光探针（ac啶橙和吡喃）进行的ΔpH测量表明，A。vinelandii NQR在各种条件下均不能转运H +。用由NQR缺陷型菌株制备的囊泡在类似测量中获得的相反结果表明NDH-1具有主要的H +泵功能。根据我们的发现，我们提出了一组简单的实验，这些实验对于明确识别细菌Na +或H +转运蛋白的泵送特异性是必要和充分的。NQR缺陷菌株，而不是NDH-1缺陷菌株，在重氮营养条件下表现出受损的生长特性，表明Na +转运在葡萄曲霉固氮中发挥作用。我们提出了一组简单的实验，这些实验对于明确鉴定细菌Na +或H +转运蛋白的泵浦特异性是必要和充分的。NQR缺陷菌株，而不是NDH-1缺陷菌株，在重氮营养条件下表现出受损的生长特性，这表明Na +转运在A. vinelandii固氮中发挥作用。我们提出了一组简单的实验，这些实验对于明确鉴定细菌Na +或H +转运蛋白的泵浦特异性是必要和充分的。NQR缺陷菌株，而不是NDH-1缺陷菌株，在重氮营养条件下表现出受损的生长特性，这表明Na +转运在A. vinelandii固氮中发挥作用。