Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.
C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Taha
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Department of Biochemistry, Michigan State University, East Lansing 48824.
Ferguson-Miller
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D000939:Epitopes; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D003576:Electron Transport Complex IV; D005022:Ethyldimethylaminopropyl Carbodiimide