Hoffmann A, Dimroth P
Mikrobiologisches Institut, Eidgenössischen Technischen Hochschule, Zürich, Switzerland.
Eur J Biochem. 1991 Mar 14;196(2):493-7. doi: 10.1111/j.1432-1033.1991.tb15841.x.
The purified ATPase of Bacillus alcalophilus (F1F0) was reconstituted into proteoliposomes by gradual removal of the detergent Triton X-100 with Amberlite XAD-2. The reconstitution was apparently highly asymmetric with nearly 100% of the F1 portion of the ATPase becoming oriented to the outside. Similar to results obtained with the soluble enzyme, the membrane-bound ATPase required Mg2+ and methanol for maximum activity. With Ca2+ or Mg2+ without methanol, 25% and 1%, respectively, of the maximum activity were observed. The ATPase was unable to pump Na+ ions but catalyzed the translocation of protons into the reconstituted proteoliposomes. Optimum proton translocation required the presence of Mg2+, not Ca2+, as divalent metal ion. The proton pump was inhibited by dicyclohexylcarbodiimide, venturicidin and NaN3. On incubation of the reconstituted ATPase with [14C]dicyclohexylcarbodiimide, subunit c of the enzyme complex became specifically labeled. The proteoliposomes catalyzed the Mg2(+)-dependent incorporation of [32P]phosphate into ATP by ATP/[32P]phosphate exchange. This exchange was little affected by monensin, but was completely abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Protons and not Na+ are thus the coupling ions of the ATPase of B. alcalophilus.
嗜碱芽孢杆菌（F1F0）的纯化ATP酶通过用Amberlite XAD - 2逐步去除去污剂Triton X - 100而被重构成蛋白脂质体。这种重构显然是高度不对称的，ATP酶的F1部分几乎100%定向于外部。与可溶性酶的结果相似，膜结合的ATP酶需要Mg2 +和甲醇以达到最大活性。在有Ca2 +或无甲醇的Mg2 +存在下，分别观察到最大活性的25%和1%。该ATP酶不能泵出Na +离子，但催化质子转运到重构的蛋白脂质体中。最佳的质子转运需要Mg2 +作为二价金属离子，而不是Ca2 +。质子泵受到二环己基碳二亚胺、venturicidin和NaN3的抑制。在用[14C]二环己基碳二亚胺孵育重构的ATP酶后，酶复合物的亚基c被特异性标记。蛋白脂质体通过ATP / [32P]磷酸盐交换催化Mg2(+)依赖的[32P]磷酸盐掺入ATP中。这种交换几乎不受莫能菌素的影响，但被解偶联剂羰基氰化物间氯苯腙完全消除。因此，质子而非Na +是嗜碱芽孢杆菌ATP酶的偶联离子。
