ESCHERICHIA-COLI F1-ATPASE SUBUNIT INTERACTIONS - BETA-SUBUNIT AND GAMMA-SUBUNIT PEPTIDES INHIBIT IN-VITRO RECONSTITUTION OF THE ACTIVE ALPHA-BETA-GAMMA COMPLEX

For biochemical analysis of subunit interactions in the proton-translo cating ATPase, a new approach with in vitro reconstitution of the Esch erichia coil alpha beta gamma complex and the peptides derived from th e subunits was established, Various portions of the beta or gamma subu nits were used for in vitro reconstitution of the alpha beta gamma com plex from the purified subunits. For the beta subunits, peptides corre sponding to residues 226-459, 254-459, and 226-365 inhibited reconstit ution, while those corresponding to residues 1-105, 1-146, and 295-459 did not. For the gamma subunits, peptides corresponding to residues 1 -192 and 74-286 exhibited inhibitory effect on reconstitution, but the peptide containing residues 191-286 did not. Only inhibitory peptides blocked the assembly of the alpha beta gamma complex which was detect ed by nondenaturing polyacrylamide gel electrophoresis. These inhibito ry peptides bound to the alpha or beta subunit on the filter, but the noninhibitory peptides did not. These results suggested that regions b eta 254-294 and gamma 74-190 have sequences important for subunit inte ractions which interfered with those in the reconstitution mixtures. B ased on comparison between X-ray crystallographic data of bovine alpha beta gamma complex and the present results, we discussed here the sig nificance of the biochemical approach adopted in this study. (C) 1997 Academic Press.