Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide.
Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.
D000180:Acrylic Resins; D000818:Animals; D002458:Cell Fractionation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006037:Glyoxal; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013816:Thermodynamics; D000099166:Polyacrylamides
D000180:Acrylic Resins; D003577:Cytochrome P-450 Enzyme System; D004800:Enzymes, Immobilized; D006037:Glyoxal; D006834:Hydrazines; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; C040388:polyacrylamide-hydrazide polymer; D000099166:Polyacrylamides; C026048:4-nitroanisole O-demethylase