Isolation of rat intestinal microsomes: Partial characterization of mucosal cytochrome P-450

Pia Lindeskog, Tapio Haaparanta, Maria Norgård, Hans Glaumann, Tiiu Hansson, Jan Åke Gustafsson

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

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 β-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.

Original languageEnglish (US)
Pages (from-to)492-501
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume244
Issue number2
DOIs
StatePublished - Feb 1 1986

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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