CYP2A3 antibody

Chuck Miller rellim at MAILHOST.TCS.TULANE.EDU
Fri Dec 4 12:29:15 EST 1998


These reports suggest antibodies against CYP2A3 are out there, but they are
not specific. Using a combination of mRNA analysis and antibody studies
might allow you to rule out the possibility of  P450s other than CYP2A3.
You might try posting your message to the Bionet P450 news group (which
specializes in this stuff) or looking at some web sites of the major
antibody vendors (e.g., http://www.researchd.com/index.htm  --not an
endorsement, I have no affiliation).

Best of results,
Chuck

Mol Carcinog 1995 Oct;14(2):130-9
Cytochrome P450 2A of nasal epithelium: regulation and role in
carcinogen metabolism.
Bereziat JC, Raffalli F, Schmezer P, Frei E, Geneste O, Lang MA
Unit of Mechanisms of Carcinogenesis, International Agency for Research on
Cancer, Lyon, France.

In this study, we found that rat nasal coumarin-7-hydroxylase (COH)
activity was two orders of magnitude higher than rat hepatic COH activity
and could be induced by adding coumarin to the rats' drinking water. In
western blot analysis, an anti-cytochrome P450 (Cyp) 2a-5 (mouse liver COH)
antibody recognized a sharp band in the microsomal fraction of rat nasal
epithelium but not of the liver; the band comigrated with Cyp2a-5. The
intensity of the band was increased by the coumarin treatment. Similarly,
in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an
mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5
mRNA; however, no hybridizable mRNA was recognized in liver preparations.
Unlike the protein level, the level of the mRNA was not increased by
coumarin. When northern blot analyses were performed with two oligoprobes
specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was
recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited
rat nasal COH activity and aflatoxin B1 (AFB1) metabolism completely. It
inhibited N-nitrosodiethylamine (NDEA) and
4-(methylnitrosamino)-1-(3-pyridyl) -1-butanone (NNK) metabolism by 80-90%.
In contrast, the hepatic metabolism of the four compounds was not affected
by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a
strong but variable inhibition of the nasal metabolism of AFB1, NDEA, and
NNK was seen. The results suggest that an enzyme or enzymes similar to
mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high
levels in rat nasal epithelium but not in the liver and that its expression
is increased by coumarin, an odorant and a substrate of Cyp2a-5. The
increase probably occurs by protein stabilization or stimulation of
translation. The results also show that the enzyme has a key role in the
nasal metabolism of three well-known carcinogens, AFB1, NDEA, and NNK and
may therefore be an important contributing factor in nasal carcinogenesis.

Biochemistry 1990 Feb 6;29(5):1322-9
The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in
human liver microsomes.
Yamano S, Tatsuno J, Gonzalez FJ
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland
20892.

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, Leu160----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 max at 450 nm. This expressed
enzyme was able to carry out coumarin 7-hydroxylation (turnover number of
15 min-1) 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. In addition, kinetics of coumarin metabolism in two
livers were monophasic over the substrate concentration tested. Km values
obtained from human liver (2.3 microM) were similar to those obtained from
lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).





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