title: Enhancement of N-hydroxy-2-aminofluorene bacterial mutagenicity by the soluble protein fraction from rat liver and partial purification of the enhancement activity.
abstract: Bacterial mutagenesis from 2-aminofluorene mediated by washed rat liver microsomes was elevated 2- to 3-fold by addition of the hepatic soluble protein fraction. Enhancement was observed at 2-aminofluorene concentrations between 1 and 20 micrograms/assay but not at 30 to 50 micrograms/assay. The soluble protein fraction (without added microsomes) did not activate 2-aminofluorene for bacterial mutagenesis. However, mutagenesis by N-hydroxy-2-aminofluorene or 2-nitrosofluorene was enhanced by the soluble protein fraction, but only when reduced nicotinamide adenine dinucleotide phosphate was also present. On the basis of chemical assay, reduced nicotinamide adenine dinucleotide phosphate reduced 2-nitrosofluorene to N-hydroxy-2-aminofluorene and protected the hydroxylamine from oxidation, thus indicating that it was the mutagenicity of N-hydroxy-2-aminofluorene (but not 2-nitrosofluorene) which was enhanced by the soluble protein fraction. Without the added soluble protein fraction, mutagenesis by N-hydroxy-2-aminofluorene or 2-nitrosofluorene was unaffected by reduced nicotinamide adenine dinucleotide phosphate. We succeeded in partially purifying a protein fraction with properties of the enhancement activity. The partially purified fraction, which represents a 14-fold increase in specific activity, was assigned a molecular weight of 33,500 by gel filtration through Sephadex G-100. This fraction was resolved into three components by polyacrylamide gel electrophoresis; the molecular weights of the three components were determined by sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis to be 33,000, 27,000, and 16,250. The mechanism of mutagenesis enhancement remains unknown.
mesh_terms: D000818:Animals; D001711:Biotransformation; D002458:Cell Fractionation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005449:Fluorenes / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D006898:Hydroxylamines / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000032:analysis / Q000378:metabolism*; D008970:Molecular Weight; D009153:Mutagens; D009249:NADP / Q000494:pharmacology; D009603:Nitroso Compounds / Q000378:metabolism / Q000633:toxicity; D011506:Proteins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics
chemical_list: D005449:Fluorenes; D006898:Hydroxylamines; D009153:Mutagens; D009603:Nitroso Compounds; D011506:Proteins; C012458:2-nitrosofluorene; D009249:NADP; C001368:N-hydroxy-2-aminofluorene