Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini.
Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.
Shin
Y
Y
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Japan.
Sawada
K
K
Nagakura
T
T
Miyanaga
M
M
Moritani
C
C
Noumi
T
T
Tsuchiya
T
T
Kanazawa
H
H
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D008024:Ligands; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D006180:Proton-Translocating ATPases; D005978:Glutathione