Document Detail

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.
MedLine Citation:
PMID:  1382585     Owner:  NLM     Status:  MEDLINE    
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)
T S Taha; S Ferguson-Miller
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  31     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1992 Sep 
Date Detail:
Created Date:  1992-10-26     Completed Date:  1992-10-26     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  9090-7     Citation Subset:  IM    
Department of Biochemistry, Michigan State University, East Lansing 48824.
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MeSH Terms
Amino Acid Sequence
Antibodies, Monoclonal / metabolism*
Antigen-Antibody Reactions
Cytochrome c Group / metabolism*
Electron Transport
Electron Transport Complex IV / metabolism*
Ethyldimethylaminopropyl Carbodiimide / analogs & derivatives,  chemistry
Macromolecular Substances
Molecular Sequence Data
Peptide Fragments / chemistry,  immunology
Peptide Mapping
Protein Binding
Grant Support
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Cytochrome c Group; 0/Epitopes; 0/Macromolecular Substances; 0/Peptide Fragments; 1892-57-5/Ethyldimethylaminopropyl Carbodiimide; 22572-40-3/1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; EC Transport Complex IV

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