Reaction of the ATPase of Escherichia coli (ECF1) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) resulted in a time- and concentration-dependent inhibition of ATPase activity. The inactivation was greatly reduced by Mg2+ ions. Close to 13 mol of EDC per mol of ECF1 was incorporated into the enzyme at 95% inhibition of ATPase activity. Two-thirds of the label was found to be associated with subunit beta with a stoichiometry of about 3 mol of EDC per mol of beta. Cleavage of EDC-modified subunit beta with cyanogen bromide and fractionation of the peptides by high-pressure liquid chromatography revealed a short segment of 33 amino acids (CB8, residues 162-194) containing 3 mol of EDC per mol of peptide. In tryptic peptide maps, two EDC-labeled fragments could be identified (T18, residues 166-183, and T20, residues 186-202). The analyses were complicated by significant internal cross-linking within the beta subunit induced by EDC. The results show that EDC modifies multiple sites in a short segment of subunit beta which includes the glutamic acids modified by dicyclohexylcarbodiimide in F1 from both E. coli and PS3. In addition to covalent modification, EDC also promoted the formation of intersubunit cross-links. The predominant cross-linked product was identified as a beta-epsilon complex by antibody binding experiments.
1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carbodiimide, inhibited ECF1-F0 ATPase activity and proton translocation through F0 when reacted with Escherichia coli membrane vesicles. The site of modification was found to be in subunit c of the F0 portion of the enzyme but did not involve Asp-61, the site labeled by the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD). EDC was not covalently incorporated into subunit c in contrast to DCCD. Instead, EDC promoted a cross-link between the C-terminal carboxyl group (Ala-79) and a near-neighbor phosphatidylethanolamine as evidenced by fragmentation of subunit c with cyanogen bromide followed by high-pressure liquid chromatography and thin-layer chromatography.
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