Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans.
The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.
Hensel
M
M
Arbeitsgruppe Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Federal Republic of Germany.
Deckers-Hebestreit
G
G
Altendorf
K
K
D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003429:Cross Reactions; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D046911:Macromolecular Substances; D012265:Ribonucleotides; D006180:Proton-Translocating ATPases