Escherichia coli F1-ATPase subunit interactions: beta and gamma subunit peptides inhibit in vitro reconstitution of the active alpha beta gamma complex.
For biochemical analysis of subunit interactions in the proton-translocating ATPase, a new approach with in vitro reconstitution of the Escherichia coli alpha beta gamma complex and the peptides derived from the subunits was established. Various portions of the beta or gamma subunits were used for in vitro reconstitution of the alpha beta gamma complex from the purified subunits. For the beta subunits, peptides corresponding to residues 226-459, 254-459, and 226-365 inhibited reconstitution, 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 detected by nondenaturing polyacrylamide gel electrophoresis. These inhibitory peptides bound to the alpha or beta subunit on the filter, but the noninhibitory peptides did not. These results suggested that regions beta 254-294 and gamma 74-190 have sequences important for subunit interactions which interfered with those in the reconstitution mixtures. Based on comparison between X-ray crystallographic data of bovine alpha beta gamma complex and the present results, we discussed here the significance of the biochemical approach adopted in this study.
Shin
Y
Y
Department of Biotechnology, Faculty of Engineering, Okayama University, Japan.
Sawada
K
K
Moritani
C
C
Kanazawa
H
H
D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011994:Recombinant Proteins
D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases