Córdoba M C, Serrano A, Córdoba F, González-Reyes J A, Navas P, Villalba J M
Departamento de Biología Celular, Universidad de Córdoba, Spain.
Biochem Biophys Res Commun. 1995 Nov 22;216(3):1054-9. doi: 10.1006/bbrc.1995.2727.
Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane.
从洋葱根中纯化得到的质膜含有两种不同的NAD(P)H脱氢酶，分子量分别为27 kDa和31 kDa，它们在物理化学性质、底物特异性和抑制剂敏感性方面存在差异。27 kDa的酶同时使用NADH和NADPH作为电子供体。31 kDa的酶对NADH具有完全特异性，并且占NADH-铁氰化物氧化还原酶的大部分。我们分别使用NADPH-和NADH-铁氰化物氧化还原酶活性作为标记，来研究27 kDa和31 kDa的酶在质膜上的取向。这些活性在通过两相分配分离得到的外翻囊泡、用去污剂Brij 58处理得到的内翻囊泡以及用Triton X-100通透处理的膜中进行测定。向外翻质膜囊泡中加入Brij 58后，NADPH-和NADH-铁氰化物氧化还原酶的激活程度与质膜H(+)-ATPase相同。在Brij 58或Triton X-100存在下测量时，氧化还原活性相似。我们的结果表明，这两种酶的催化位点都朝向质膜的细胞质侧。
