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Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450
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 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.
Insights
This study details a rat small intestine cell isolation method, yielding subcellular fractions. Researchers identified specific cytochrome P-450 isozymes in these fractions using enzyme assays and Western blotting.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Investigating subcellular fractions of rat small intestinal mucosal cells is crucial for understanding drug metabolism and cellular processes.
- Accurate isolation and characterization of these fractions are essential for reliable biochemical analysis.
Purpose of the Study:
- To develop and validate a procedure for isolating subcellular fractions from rat small intestinal mucosal cells.
- To characterize these fractions using marker enzymes and identify specific cytochrome P-450 isozymes.
Main Methods:
- Rat small intestinal mucosal cells were harvested by scraping and homogenized using a Potter-Elvehjem device followed by sonication.
- Differential centrifugation was employed to isolate subcellular fractions (10,000g pellet, 105,000g pellet/microsomal fraction, supernatant).
- Marker enzyme activities (7-ethoxyresorufin-O-deethylase, NADPH-cytochrome c reductase) and cytochrome P-450 isozymes were analyzed using antibody inhibition, Western blotting, and peroxidase-antiperoxidase methods.
Main Results:
- Optimal yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet.
- Anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies significantly inhibited 7-ethoxyresorufin O-deethylation, indicating the presence of BNF-inducible isozymes.
- Western blotting confirmed the presence of BNF-inducible isozymes in both BNF-treated and untreated rats, with localization studies showing general villus staining after BNF treatment.
Conclusions:
- The described procedure effectively isolates subcellular fractions from rat small intestinal mucosal cells.
- Specific cytochrome P-450 isozymes, particularly BNF-inducible forms, are present in rat intestinal microsomes.
- These findings provide a foundation for further studies on intestinal drug metabolism and the role of cytochrome P-450 in this tissue.