Tokuda H, Unemoto T
J Biol Chem. 1984 Jun 25;259(12):7785-90.
The coupling site of the Na+ pump to the respiratory chain of Vibrio alginolyticus was examined using membrane fractions prepared from the wild type, Na+ pump-deficient mutants, and spontaneous revertant. NADH oxidase of the wild type and revertant specifically required NA+ for maximum activity, whereas Na+ was not essential for the NADH oxidase of mutants. Similar to the Na+ pump in whole cells, the Na+-dependent NADH oxidase in membranes had a pH optimum in the alkaline region. A respiratory inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), inhibited the Na+-dependent NADH oxidase but had little effect on the NA+-independent activity of mutant membranes. NADH:quinone oxidoreductase was found to be the Na+-dependent HQNO-sensitive site of the NADH oxidase. In the wild type cells, HQNO was also found to cause a strong inhibition of the Na+ pump with little effect on the overall H+ extrusion by respiration. The inhibition of the Na+ pump by HQNO was overcome by oxidized, but not reduced, N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD). In the presence of oxidised TMPD, the electron flow NADH to oxygen seemed to bypass the HQNO-sensitive site and energize the Na+ pump. From these results, it was concluded that the Na+ pump is coupled to the respiratory chain at the step of NADH:quinone oxidoreductase.
利用从野生型、钠泵缺陷型突变体和自发回复突变体中制备的膜组分，研究了溶藻弧菌钠泵与呼吸链的偶联位点。野生型和回复突变体的NADH氧化酶的最大活性特别需要钠离子，而钠离子对于突变体的NADH氧化酶并非必需。与全细胞中的钠泵类似，膜中依赖钠离子的NADH氧化酶在碱性区域具有最适pH值。呼吸抑制剂2-庚基-4-羟基喹啉-N-氧化物（HQNO）抑制依赖钠离子的NADH氧化酶，但对突变体膜的不依赖钠离子的活性影响很小。发现NADH：醌氧化还原酶是NADH氧化酶中依赖钠离子且对HQNO敏感的位点。在野生型细胞中，还发现HQNO对钠泵有强烈抑制作用，而对呼吸作用引起的整体氢离子外排影响很小。HQNO对钠泵的抑制作用可被氧化型而非还原型的N，N，N'，N'-四甲基对苯二胺（TMPD）克服。在氧化型TMPD存在下，从NADH到氧气的电子流似乎绕过了对HQNO敏感的位点并为钠泵供能。从这些结果得出结论，钠泵在NADH：醌氧化还原酶步骤与呼吸链偶联。
