Groth G, Walker J E
Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):351-7. doi: 10.1042/bj3180351.
A highly purified and monodisperse preparation of proton-translocating F1F0-ATPase from bovine heart mitochondria is an assembly of 16 unlike polypeptides. This preparation has been reconstituted in the presence of various detergents into unilamellar phospholipid vesicles. Incorporation of the enzyme into vesicles increases the ATP hydrolase activity of the enzyme by 10-20-fold, depending on the detergent, and the highest activities of ATP hydrolysis, 70 units/mg, were obtained by reconstitution from dodecylmaltoside or CHAPS. This activity is mostly sensitive to inhibitors that act on the F0 membrane sector of the complex. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-methoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co-reconstitution of the enzyme with bacteriorhodopsin, it was demonstrated that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteristic biological functions of the F1F0-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its physical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.
一种来自牛心线粒体的高度纯化且单分散的质子转运F1F0 - ATP酶制剂是由16种不同多肽组成的装配体。该制剂已在各种去污剂存在的情况下重构成单层磷脂囊泡。将该酶掺入囊泡中会使酶的ATP水解酶活性提高10至20倍，这取决于去污剂，通过从十二烷基麦芽糖苷或CHAPS进行重构可获得最高的ATP水解活性，即70单位/毫克。这种活性对作用于该复合物F0膜部分的抑制剂最为敏感。通过对pH敏感探针9 - 氨基 - 6 - 氯 - 2 - 甲氧基吖啶的猝灭表明，重构后的酶能够以ATP依赖的方式形成跨膜质子梯度。通过将该酶与细菌视紫红质共同重构，证明在光诱导质子梯度存在的情况下，该酶能够从ADP和磷酸盐合成ATP。因此，利用纯化的酶已证明了线粒体中F1F0 - ATP酶的特征性生物学功能。因此，就其物理和生化性质而言纯化的酶满足了形成二维和三维晶体的重要前提条件。
