Hilpert W, Dimroth P
Eur J Biochem. 1984 Feb 1;138(3):579-83. doi: 10.1111/j.1432-1033.1984.tb07953.x.
Methylmalonyl-CoA decarboxylase from Veillonella alcalescens which was isolated by affinity chromatography on avidin-Sepharose was incorporated into phospholipid vesicles by the detergent dilution method with octyl glucoside as the detergent. By this procedure the Na+ pump activity was reconstituted. The optimal octyl glucoside concentration for reconstitution was about 2.8%. The activity of reconstituted Na+ transport increased with the amount of enzyme present during reconstitution until a plateau was reached at about 7 micrograms enzyme/mg phospholipid. All four polypeptides of the decarboxylase were incorporated into the proteoliposomes but the relative amounts of alpha and gamma subunits were considerably reduced. The reconstitution process was highly asymmetric, since about 80% of the decarboxylase was oriented in the proteoliposomes with the substrate binding site facing to the interior. The orientation was determined from the increase of methylmalonyl-CoA decarboxylase activity by destruction of the membrane permeability barrier. It was also deduced from the amount of enzyme which was not accessible from the outside to inactivation by avidin. With these reconstituted vesicles, a steady-state internal Na+ concentration was established by methylmalonyl-CoA decarboxylation which under optimized conditions was about 30-fold higher than in the incubation medium. Sodium ion accumulation in the presence of the Na+-carrying ionophores nigericin or monensin was practically nil. In the presence of valinomycin or carbonylcyanide-p-trifluoromethoxy phenylhydrazone the rate of Na+ transport and its steady-state internal concentration were considerably higher than in the controls which is consistent with the function of methylmalonyl-CoA decarboxylase as an electrogenic Na+ pump.
通过抗生物素蛋白-琼脂糖亲和层析分离得到的产碱韦荣球菌甲基丙二酰辅酶A脱羧酶，采用以辛基葡糖苷为去污剂的去污剂稀释法将其整合到磷脂囊泡中。通过该方法重建了Na⁺泵活性。重建的最佳辛基葡糖苷浓度约为2.8%。重建的Na⁺转运活性随着重建过程中存在的酶量增加而增加，直到在约7微克酶/毫克磷脂时达到平台期。脱羧酶的所有四种多肽都整合到了蛋白脂质体中，但α和γ亚基的相对量显著减少。重建过程高度不对称，因为约80%的脱羧酶以底物结合位点面向内部的方式定向在蛋白脂质体中。该方向是通过破坏膜通透性屏障后甲基丙二酰辅酶A脱羧酶活性的增加来确定的。这也可以从外部无法接触到的、因抗生物素蛋白而失活的酶量推断出来。利用这些重建的囊泡，通过甲基丙二酰辅酶A脱羧作用建立了稳定状态的内部Na⁺浓度，在优化条件下，该浓度比孵育培养基中的浓度高约30倍。在携带Na⁺的离子载体尼日利亚菌素或莫能菌素存在的情况下，Na⁺积累几乎为零。在缬氨霉素或羰基氰化物-对-三氟甲氧基苯腙存在的情况下，Na⁺转运速率及其稳定状态的内部浓度比对照高得多，这与甲基丙二酰辅酶A脱羧酶作为电生Na⁺泵的功能一致。
