Effect of immobilization on the activity of rat hepatic microsomal cytochrome P450 enzymes.
Cytochrome P450 enzyme preparations were obtained from phenobarbital-treated rats. On immobilization in Romicon PM 10 polysulfone anisotropic hollow fibers, 20-25% of the original activity remained. The immobilization procedure did not significantly alter the Km of the preparations when 7-ethoxycoumarin was the substrate. Coimmobilization of the preparations with glucose-6-phosphate dehydrogenase allowed cofactor recycling to take place but resulted in an apparent drop in the value for the Km. The Vmax of immobilized preparations with or without cofactor recycling was about 25% of that observed with the nonimmobilized preparations. The flow rate through the reactors greatly affected the time at which product was detected in the effluent from the reactor and the amount of 7-hydroxycoumarin produced. It is suggested that this reflects alternative fates of the NADPH cofactor when residence times in the reactor are lengthened. Increasing the amounts of protein immobilized increased the Km and decreased the Vmax values in comparison to the free enzyme. Immobilization increased the stability of preparations with respect to ionic strength and storage. Little effect was noted with regard to changes in temperature stability or pH optima of immobilized preparations.
Fernandez-Salguero
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Biological Laboratory, University of Kent, Canterbury, UK.
Gutierrez-Merino
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Bunch
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D000818:Animals; D001711:Biotransformation; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism; D004800:Enzymes, Immobilized; D008297:Male; D008567:Membranes, Artificial; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D017208:Rats, Wistar; D014468:Umbelliferones / Q000378:metabolism
D003374:Coumarins; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital