RECONSTITUTION OF THE F1-ATPASE ACTIVITY FROM PURIFIED ALPHA-SUBUNIT,BETA-SUBUNIT, GAMMA-SUBUNIT AND DELTA-SUBUNIT OR EPSILON-SUBUNIT WITHGLUTATHIONE-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 alp ha and beta subunits recovered as soluble form were purified by hydrox yapatite column chromatography. Since the gamma subunit was overexpres sed 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 renatura tion, the active gamma subunit for reconstitution of the F-1-ATPase ac tivity with the purified alpha and beta subunit was obtained. The delt a and epsilon subunits which were fused to the carboxy terminus of glu tathione S-transferase (GST) were overproduced and purified by affinit y chromatography. These fused proteins (delta-GST and epsilon-GST) wer e incubated with the purified alpha, beta and gamma subunits and appli ed to affinity chromatography. The alpha beta gamma delta-GST and alph a beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectivel y, with a subunit stoichiometry similar to that in the native F-1-ATPa se, indicating that active complexes could be reconstituted with the f used proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the a ctive complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta GST bound the gamma subunit s ignificantly, and the alpha and beta subunits very weakly.