The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes
- National Institutes of Health, Bethesda, MD (USA)
Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a {lambda}gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu{sup 160}{yields}His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a {lambda}{sub max} at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIa mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. These data establish that the CYP2A3 gene product is primarily responsible for coumarin 7-hydroxylase activity in human liver. The level of expression of this activity varied up to 40-fold between livers. Levels of IIA mRNA also varied significantly between liver specimens, and three specimens had no detectable mRNA.
- OSTI ID:
- 6709401
- Journal Information:
- Biochemistry; (USA), Vol. 29:5; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
Similar Records
Molecular cloning and nucleotide sequence of cDNA for human liver arginase
Monoclonal antibody-directed phenotyping of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues
Related Subjects
HYDROXYLASES
AMINO ACID SEQUENCE
LIVER
ENZYME ACTIVITY
RECOMBINANT DNA
DNA SEQUENCING
ANTIBODIES
COUMARIN
HISTIDINE
LEUCINE
MAN
MESSENGER-RNA
MICROSOMES
VACCINIA VIRUS
AMINO ACIDS
ANIMALS
AZOLES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
DIGESTIVE SYSTEM
DNA
ENZYMES
ESTERS
GLANDS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
LACTONES
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANOIDS
ORGANS
OXIDOREDUCTASES
PARASITES
PRIMATES
PYRANS
RIBOSOMES
RNA
STRUCTURAL CHEMICAL ANALYSIS
VERTEBRATES
VIRUSES
550201* - Biochemistry- Tracer Techniques