Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450.
A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology; D005260:Female; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D008861:Microsomes / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000032:analysis; D010088:Oxidoreductases / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D009245:NADH Dehydrogenase