Hicks D B, Krulwich T A
Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York 10029.
J Biol Chem. 1990 Nov 25;265(33):20547-54.
The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.
嗜碱芽孢杆菌OF4的F1F0 - ATP合酶以具有重组活性的形式被纯化出来，产量可观，且在适当激活时具有较高的特异性ATP酶活性。纯化过程包括在20%甘油和大豆卵磷脂存在的情况下，用辛基葡糖苷提取洗涤过的膜泡，随后进行硫酸铵分级分离和蔗糖密度梯度离心。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，纯化后的制剂被分离成七条带，分别对应F1的五个亚基α、β、γ、δ和ε，以及F0的b和c亚基。二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶分析揭示了F0的α亚基的一个候选物。嗜碱芽孢杆菌OF4 F1F0的MgATP酶活性几乎检测不到，但可被亚硫酸盐、甲醇和硫代辛基葡糖苷最佳地刺激100倍以上。该酶被N，N'-二环己基碳二亚胺和叠氮化钠抑制，但不被大肠杆菌F1的抑制剂金精三羧酸抑制。重组到蛋白脂质体中的F1F0催化ATP酶活性、ATP - Pi交换以及ATP依赖的ΔpH和Δψ形成。质子载体刺激ATP水解，而其他活性则被质子载体消除。这些活性既不依赖于添加的钠离子，也不受其显著影响。由还含有Na⁺/H⁺反向转运蛋白的粗辛基葡糖苷提取物制成的F1F0蛋白脂质体被证明可催化ATP依赖的Na⁺摄取，该摄取对羰基氰化物间氯苯腙完全敏感；在含有更纯化但缺乏Na⁺/H⁺反向转运蛋白的F1F0的蛋白脂质体中不存在Na⁺摄取活性。这些数据表明，F1F0转运质子，并且在能量偶联中不会用Na⁺替代H⁺。
