Immobilization of pig liver microsomes |
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Authors: | Magda Ibrahim Martine Decolin Anne-Marie Batt Edith Dellacherie Gerard Siest |
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Institution: | (1) Laboratoire de Biochimie Pharmacologique, Unité Associée au CNRS Nℴ 597, Centre du Médicament, 30 rue Lionnois, 54000 Nancy, France;(2) Laboratoire de Chimie Physique Macromoléculaire, Equipe Associée au CNRS Nℴ 494, ENSIC, 1 rue Grandville, 54042 Nancy Cédex, France |
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Abstract: | Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1’-carbonyldiimidazole.
Microsomes were also entrapped inside Ca-alginate andk-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity
of the monooxygenase system was demonstrated by theN-demethylation of aminopyrine, theO-demethylation ofp-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30–40% and a 60–70% decrease in V
max
app
for theOandN-demethylations were respectively observed. The V
max
app
values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for
the freely suspended microsomal cytochrome P-450.
Under storage at 4°C, microsomes entrapped insidek-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose
improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized
microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped insidek-carrageenan or Ca-alginate can be used to follow up the continuous metabolization ofp-nitroanisole for several hours in a stirred-batch reactor. |
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Keywords: | Cytochrome P-450 microsomes entrapment of microsomes covalent binding pig liver |
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