Subunits of the H+-ATPase of Escherichia coli. Overproduction of an eight-subunit F1F0-ATPase following induction of a lambda-transducing phage carrying the unc operon.

The inhibitory effect of dicyclohexylcarbodiimide (DCCD) on the activity of the adenosine-triphosphatase of Escherichia coli (ECF1) has been examined in detail. DCCD reacted with ECF1 predominantly in beta subunits with a maximum of 2 mol of reagent per mole of ECF1 being incorporated in these subunits. Ninety-five percent inhibition of steady-state or multistate ATPase activity required incorporation of 1 mol of DCCD per mole of enzyme into beta subunits. Seventy-five percent inhibition of the initial rate of unisite catalysis was only obtained after incorporation of 2 mol of DCCD per mole of ECF1 into beta subunits. Analyses of the kinetics of unisite catalysis and nucleotide binding experiments both indicate that DCCD binds outside the substrate ATP binding site. Inhibition by this reagent appears to be due in part to an effect on the catalytic sites but mainly to the blocking of cooperativity between these sites.

Section: Methodsmentioning

confidence: 99%

Effect of dicyclohexylcarbodiimide on unisite and multisite catalytic activities of the adenosine triphosphatase of Escherichia coli

Tommasino¹,

Capaldi²

1985

“…The data shown in Figure 1 were obtained with membrane vesicles from E. coli prepared by the French press method of Foster et al (1980). Such membranes are thought to be insideout in orientation with respect to the intact bacterium.…”

Section: Resultsmentioning

confidence: 99%

“…Membranes, ECF^Fq, and ECF! were each prepared from this strain after induction of the X phage as described by Foster et al (1980) and Foster & Fillingame (1979). ECF,-stripped membranes were prepared by incubating membranes of 5 mg of protein/mL in 1 mM tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 1 mM dithiothreitol, 0.5 mM EDTA, and 5% glycerol for 1 h at 25 °C.…”

mentioning

confidence: 99%

Modification of the F0 portion of ECF1-F0 by the water-soluble carbodiimide EDC and effect on the proton channeling function

Lotscher¹,

DeJong²,

Capaldi³

1984

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.

“…Source of Cells and Preparation of ECF x. E. coli strain KY 7485 containing a X-transducing phage which carries all eight ATPase genes of the unc operon was kindly provided by Dr. R. H. Fillingame, University of Wisconsin, Madison, WI. The F, complex was purified from this strain after induction of the X phage (Foster et al, 1980) according to the procedure described by Foster & Fillingame (1979). Protein was determined according to the procedure described by Lowry et al (1951).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of ATPase activity of E. coli F1 by the water-soluble carbodiimide EDC is due to modification of several carboxyls in the .beta. subunit

Lotscher¹,

DeJong²,

Capaldi³

1984

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.