Purification and photoaffinity labelling of a rat cytosolic binding protein specific for 3-methylcholanthrene

Abstract

Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.

A novel 4 S [3H]β-naphthoflavone-binding protein in liver cytosol of female Sprague–Dawley rats treated with aryl hydrocarbon receptor agonists

“…The latter does not appear to be induced by PAHs since, in the livers of 3-MC-treated rats, the specific [$H]B[a]P binding to a 4 S fraction was not increased [40] and the cytoplasmic 4 S [$H]3-MC-binding protein was transferred to nuclei without an apparent increase in its overall content [39]. The molecular mass of the 4 S [$H]β-NF-binding protein from β-NF-treated rats was $ 42 kDa ( Figure 5A), and thus was similar to that of the 4 S PAH-binding protein determined with [$H]3-MC ($ 39 kDa) [38] or [$H]B[a]P ($ 40 kDa) [18] as ligands. Since the [$H]β-NF-binding protein was eluted with the void volume of Sephacryl S-200 gel filtrate before the radioligand binding, but was eluted as a 42 kDa component after binding of [$H]β-NF, it appears that the large complex or aggregate disaggregates upon binding to yield the 42 kDa protein ( Figure 5A).…”

“…β-NF was shown to be a high-affinity ligand for a constitutive 4 S PAH-binding protein because it significantly blocked the specific binding of [$H]3-MC [5,20] and that of [$H]B[a]P [21] in liver cytosols of untreated male SD rats. Hence, these rats (either of unknown source, or CD rats from Charles River, Margate, Kent, U.K.) as well as male Wistar [38,39] and SD rats from Sasco (Omaha, NE, U.S.A.) [11,12,40] are considered to be ' 4-Spositive '. By contrast, binding of [$H]B[a]P to a 4 S protein was undetectable in male Harlan SD rats from Houston, TX, U.S.A. (see [12]), which were thus considered to be ' 4-S-negative ' [12].…”