Document Detail

Cytochrome b5, cytochrome c, and cytochrome P-450 interactions with NADPH-cytochrome P-450 reductase in phospholipid vesicles.
MedLine Citation:
PMID:  2847775     Owner:  NLM     Status:  MEDLINE    
Upon incubation of detergent-solubilized NADPH-cytochrome P-450 reductase and either cytochrome b5 or cytochrome c in the presence of a water-soluble carbodiimide, a 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), covalently cross-linked complex was formed. The cross-linked derivative was a heterodimer consisting of one molecule each of flavoprotein and cytochrome, and it was purified to 90% or more homogeneity. The binary covalent complex between the flavoprotein and cytochrome b5 was exclusively observed following incubation of all three proteins including NADPH-cytochrome P-450 reductase, cytochrome b5, and cytochrome c in L-alpha-dimyristoylphosphatidylcholine vesicles, and no heterotrimer could be identified. The isolated reductase-cytochrome b5 complex was incapable of covalent binding with cytochrome c in the presence of EDC. No clear band for covalent complex formation between PB-1 and reductase was seen with the present EDC cross-linking technique. More than 90% of the cross-linked cytochrome c in the purified derivative was rapidly reduced upon addition of an NADPH-generating system, whereas approximately 80% of the cross-linked cytochrome b5 was rapidly reduced. These results showed that in the greater part of the complexes, the flavin-mediated pathway for reduction of cytochrome c or cytochrome b5 by pyridine nucleotide was intact. When reconstituted into phospholipid vesicles, the purified amphipathic derivative could hardly reduce exogenously added cytochrome c, cytochrome b5, or PB-1, indicating that the cross-linked cytochrome shields the single-electron-transferring interface of the flavoprotein. These results suggest that the covalent cross-linked derivative is a valid model of the noncovalent functional electron-transfer complex.
Y Nisimoto; H Otsuka-Murakami
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Publication Detail:
Type:  In Vitro; Journal Article    
Journal Detail:
Title:  Biochemistry     Volume:  27     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1988 Aug 
Date Detail:
Created Date:  1989-01-09     Completed Date:  1989-01-09     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5869-76     Citation Subset:  IM    
Department of Biochemistry, Aichi Medical University, Japan.
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MeSH Terms
Cross-Linking Reagents
Cytochrome P-450 Enzyme System / metabolism
Cytochrome b Group / metabolism
Cytochrome c Group / metabolism
Cytochromes b5
Electron Transport
Ethyldimethylaminopropyl Carbodiimide
Flavin Mononucleotide / metabolism
Microsomes, Liver / enzymology
NADPH-Ferrihemoprotein Reductase / metabolism*
Reg. No./Substance:
0/Cross-Linking Reagents; 0/Cytochrome b Group; 0/Cytochrome c Group; 0/Liposomes; 146-17-8/Flavin Mononucleotide; 1892-57-5/Ethyldimethylaminopropyl Carbodiimide; 9035-39-6/Cytochromes b5; 9035-51-2/Cytochrome P-450 Enzyme System; EC Reductase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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