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At least 19 recordsLinked to original sources
PubMed1984

Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function.

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.

Carbodiimides↗
PubMed1984

Inhibition of the adenosinetriphosphatase activity of Escherichia coli F1 by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is due to modification of several carboxyls in the beta subunit.

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.

Amino Acids↗
PubMed1986

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.

Acetylglucosamine↗
PubMed1976

The role of the carbodiimide-reactive component of the adenosine-5'-triphosphatase complex in the proton permeability of Escherichia coli membrane vesicles.

Membrane vesicles isolated from wild-type and dicyclohexylcarbodiimide-resistant strains of Escherichia coli exhibit identical respiration-dependent transport activities, and in both cases, this activity is abolished by extraction of the vesicles with 1.0 M guanidine-HCl. Transport activity of extracted wild-type vesicles is completely restored by exposing the vesicles to lipophilic or water-soluble carbodiimides, while transport activity of the mutant vesicles is not restored by exposure to lipophilic carbodiimides. Strikingly, however, complete reactivation of transport in mutant vesicles is observed with water-soluble carbodiimides. Similarly, the Ca2+, Mg2+-stimulated ATPase activity of wild-type vesicles is inhibited by both classes of carbodiimides, while the ATPase activity of mutant vesicles is inhibited by water-soluble carbodiimides, but resistant to inhibition by lipophilic carbodiimides. The carbodiimide-reactive component of the membraneous Ca2+, Mg2+-stimulated ATPase complex in wildtype vesicles is readily labeled with N,N'-dicyclohexyl[14C]-carbodiimide, while the analogous component in mutant vesicles is not reactive. Alternatively, when vesicles are treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [14C]methiodide, a water-soluble carbodiimide, the carbodiimide-reactive component is labeled to a similar degree in both preparations. The results suggest that the altered carbodiimide-reactive proteolipid in the dicyclohexylcarbodiimide-resistant mutant is specifically defective in its ability to react with lipophilic carbodiimides. In addition, these and other findings indicate that the increase in proton permeability observed on extraction of isolated membrane vesicles with chaotropic agents is due exclusively to an effect on the carbodiimide-reactive component of the Ca2+, Mg2+-stimulated ATPase complex.

Adenosine Triphosphatases↗
PubMed1986

Binding of DNA to albumin and transferrin modified by treatment with water-soluble carbodiimides.

N-Acylurea derivatives of albumin and transferrin prepared with the water-soluble carbodiimides N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide and N-ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide have been found to bind different types of DNA. The two proteins were reacted with varying amounts of carbodiimide in water at pH 5.5 for 36-60 hr at 20 degrees, and then purified. In the case of iron-loaded transferrin, reactions with carbodiimides were in phosphate-buffered saline (pH 7.5) to prevent loss of iron from the protein. [3H]N-Ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the modified proteins, and gel electrophoresis for changes in charge and possible aggregation through cross-linking. Binding of DNA to N-acylurea proteins was studied by means of gel electrophoresis and nitrocellulose filter binding. N-Acylurea albumin and N-acylurea transferrin at low concentrations retarded the migration of lambda-Pstl restriction fragments, pBR322 plasmid and M13 mp8 single-stranded DNA on agarose gels, while at higher concentrations of modified protein the N-acylurea protein-DNA complexes were unable to enter the gel. Nitrocellulose filter assays showed that binding pBR322 DNA and calf thymus DNA to N-acylurea proteins is rapid and dependent on protein concentration and the ionic strength of the medium. N-Acylurea albumins prepared with each each of the two carbodiimides gave comparable plots for DNA bound versus protein concentration. On the other hand, binding of DNA by N-acylurea transferrins differed according to the carbodiimide used in the synthesis. N-Acylurea CDI-tkransferrin (prepared with tertiary carbodiimide) was less effective than either of the two N-acylurea albumins in binding DNA. In contrast with these results, N-acylurea Me+-CDI-transferrin (prepared with quaternary carbodiimide) was far more effective in binding DNA and in this respect was similar to the N-acylurea albumins. On the basis of experiments in which N-acylurea protein-DNA complexes were treated with heparin, two types of binding could be distinguished. These were a weak binding occurring in the initial stages of interaction and a tight binding which developed on further incubation of the complexes. These studies show that binding of DNA by N-acylurea proteins is a reversible process dependent on ionic strength; interaction appears to be electrostatic in nature, although other forms of binding might be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗