Dergunov A D, Zhukova I G, Nikul'tseva T P, Degteva G K, Nosova T V
Biokhimiia. 1982 Mar;47(3):478-88.
The NADH-dehydrogenase isolated from the M. lysodeikticus membranes was reconstituted into liposomes from the lipids obtained from the same membranes. The presence and degree of the reconstitution were investigated by two-dimensional immunoelectrophoresis and photoreactive hydrophobic label. The quenching of protein fluorescence by the aqueous quencher J- was practically the same for the enzyme in the reconstituted system and in the detergent solution, whereas the quencher interacting with the membrane--cetylpyridinium chloride--was effective in the first case and not effective in the second one. Evidence for the energy transfer from protein chromophores of NADH dehydrogenase in the proteoliposomes (lambda excit = 286 nm) to the hydrophobic fluorescent probe pyrene was obtained. It was found that about 30% of the chromophores in the enzyme molecule are involved in this process. The hydrophobic spin probe, whose paramagnetic fragment is located on the surface and not inside the hydrophobic phase of the membrane, can act as electron acceptor during NADH oxidation in the reconstituted system. The data obtained are suggestive of the exposure of the bulk of the enzyme molecule to the environment and of interaction of the smaller part of the molecule with the lipid phase. The active center is located on the part of the enzyme molecule which is exposed to water. It is assumed that the NADH-dehydrogenase molecule is exposed to water. It is assumed that the NADH-dehydrogenase molecule is involved in heat diffusion which facilitates the active center interaction with the membrane surface.
从溶壁微球菌膜中分离出的NADH脱氢酶，用从同一膜中获得的脂质重构成脂质体。通过二维免疫电泳和光反应性疏水标记研究了重构的存在情况和程度。对于重构体系中的酶和去污剂溶液中的酶，水性猝灭剂J-对蛋白质荧光的猝灭作用几乎相同，而与膜相互作用的猝灭剂十六烷基吡啶氯化物在第一种情况下有效，在第二种情况下无效。获得了从蛋白脂质体中NADH脱氢酶的蛋白质发色团（激发波长λ = 286 nm）到疏水荧光探针芘的能量转移的证据。发现酶分子中约30%的发色团参与了这一过程。在重构体系中，其顺磁片段位于膜表面而非疏水相内部的疏水自旋探针，可在NADH氧化过程中充当电子受体。所获得的数据表明，酶分子的大部分暴露于环境中，且分子的较小部分与脂质相相互作用。活性中心位于酶分子暴露于水的部分。假定NADH脱氢酶分子暴露于水。假定NADH脱氢酶分子参与热扩散，这有利于活性中心与膜表面的相互作用。
