PURIFICATION OF THE H+-ATPASE FROM RHODOBACTER-CAPSULATUS, IDENTIFICATION OF THE F1F0 COMPONENTS AND RECONSTITUTION OF THE ACTIVE ENZYME

作     者：GABELLINI, N GAO, Z ECKERSKORN, C LOTTSPEICH, F OESTERHELT, D 

作者机构：Max-Planck-Institut für Biochemie Martinsried F.R.G. 

出 版 物：《BIOCHIMICA ET BIOPHYSICA ACTA》 (Biochim. Biophys. Acta Bioenerg.)

年 卷 期：1988年第934卷第2期

页      面：227-234页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

主　　题：ATPase purification, H + - F 0 F 1 Enzyme reconstitution ( Rb. capsulatus ) SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis DCCD N , N ′-dicyclohexylcarbodiimide FCCP carbonylcyanide p -(trifluoromethoxy)-phenylhydrazone octylglucoside n -octyl-β-D-glucopyranoside Triton X-100 octylphenoxypoly(ethoxyethanol) Tricine N -[2-hydroxy-1,1-(bis(methyl)ethyl]glycine DTT dithiothreitol EDTA ethylene dinitrile tetra acetic acid HPLC high performance liquid chromatography P i inorganic phosphate 

摘      要：A simple method has been adopted for the isolation of the H+-ATPase from cell membranes of Rhodobacter capsulatus that is based on octylglucoside solubilization. The highly purified F0F1 enzyme catalyzes ATP hydrolysis and DCCD-sensitive ATP-Pi exchange. The isolated complex consists of nine different polypeptides with apparent molecular masses of 50, 45, 34, 29, 23, 20, 18.5, 11 and 7.5 kDa. The N-terminal sequence of the eight largest subunits were determined by automated Edman degradation. The five F1 polypeptides could be readily identified by their homology to the F1 subunits .alpha., .beta., .gamma., .delta. and .epsilon. from other Rhodospirillaceae. In contrast to prokaryotic F0 complexes known today, the membrane portion of H+-ATPase from R. capsulatus was found to be composed of four polypeptides. Three of these correspond to subunit a, b and c of the Escherichia coli enzyme (29, 18.5 and 7.5 kDa). A fourth subunit of 23 kDa may be also related to subunit b. The ATP-synthase of this photosynthetic bacterium is in this respect similar to that of chloroplasts.