Naphthalene is an environmenlal contaminanl whi(h causes selective toxi('it.v 1o routine (:lara (:ells at low doses. Naphthalene-induced Clara (:ell loxicity (;orrelales with the presence of c!/p 2f2, the putative enzyme responsible for the stereospecific melabolism ()[' naphthahme to I R,2S naphthalene oxide. The finding of an orthologous (mzvme in human lung (CYP2F1) supports the need to und('rsland the melaboli(: and kinetic differences in the aclivation of lung toxicants. To produ(:e sufficient amounts of mouse cyp 2f2 for such sludies, we have embarked on the expression of the eDNA fl)r cyp 2f2using the baculovirus exl)ression svslem, l'h' (I)NA nncleotide sequence, fully sequenced in both directions, demonstrated the authenticity of the sequence used. S1)S PA(',E analysis of whole cell lvsaws from Tn5 insect (:ells infected with our recom binant baculovirus, u[)fm ('oomasie blue staining, showed tile over-expression of a 50 kDa pro|ein. ['xpl'ession levels of lifts protein haw been estimated as being 2()c/( of Iotal cellular protein. Western blot analysis using cyp 2f2 polyclonal antibodies gaw a strong signal with the 50 kDa protein thus con firming its identiD" as cyp 2f2. t)450 spectral analysis of the recombinant enzyme produced in "ln5 c(qls, supplemenled with exogenous heroin during expression, yielded a peak at .153 illll corresponding to expression levels of approximately 0.3 nmoles P 150 per 35 mm culture dish. Incubations of Tn5 ('ell lysate preparations ('ont aining recombinant cyp 2f2 with NADPtt ytochrome P450 reductase and NA l)Ptt melabolized naphthalene stereospe(ifi(:ally o tho 1R, 2S-epoxide.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology