Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse lives

Kaipainen, Pekka, Nebert, Daniel W. and Lang, Matti A. (1984) Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse lives. European Journal of Biochemistry, 144 3: 425-431. doi:10.1111/j.1432-1033.1984.tb08483.x


Author Kaipainen, Pekka
Nebert, Daniel W.
Lang, Matti A.
Title Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse lives
Journal name European Journal of Biochemistry   Check publisher's open access policy
ISSN 0014-2956
1432-1033
Publication date 1984-11
Sub-type Article (original research)
DOI 10.1111/j.1432-1033.1984.tb08483.x
Volume 144
Issue 3
Start page 425
End page 431
Total pages 7
Place of publication Oxford, United Kingdom
Publisher Wiley-Blackwell Publishing
Language eng
Formatted abstract
Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome · CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated ‘P-450Coh’.
     Anti-(P-450coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 ± 100 nucleotides.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: National Research Centre for Environmental Toxicology Publications
 
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Created: Mon, 30 Nov 2009, 15:03:16 EST