ISSN:
1432-1912
Keywords:
Cytochrome P-450
;
Metyrapone
;
Active Centers of Enzyme
;
Induction
;
Enzyme Inhibition
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Summary Metyrapone inhibits the N-demethylation of aminopyrine and ethylmorphine in rat liver microsomes non-competitively with concentrations in the micromolar range, and competitively in the range of 10−4 M. In the case of non-competitive inhibition, enzyme and inhibitor are present in similar amounts (“mutual depletion system”). Under these conditions Lineweaver-Burk kinetics do not apply, since the portion of inhibitor bound to the enzyme cannot be neglected. Inhibition of microsomal N-demethylating activity by increasing amounts of metyrapone and application of equations required for a mutual depletion system enable us to determine the following kinetic parameters: (1) the concentration of catalytically active and metyrapone sensitive centers (E t ) of cytochrome P-450, (2) their turnover number (TN), and (3) the true dissociation constant of the enzyme inhibitor complex, K i .—With aminopyrine as substrate and microsomes from phenobarbital (PB) pretreated rats, 1 mg of protein contains 4.7 nmoles of E t , i.e. about 2.2 catalytically active sites per molecule of cytochrome P-450; TN=3.4 min−1, and K i =2.2·10−6 M. The corresponding values for ethylmorphine and control rats are: E t =3.1 nmolles per mg of protein (4.4 sites per molecule), TN=1.94 min−1; K i =2.37·10−6M. PB-pretreated rats: E t =7.3 nmoles per mg of protein (3.5 sites per molecule) TN=2.0 min−1; K i =2.3·10−6 M. Comparing the values obtained for PB-pretreated rats and controls reveals that K i and TN remain constant but E t rises 2.3 fold. This is regarded to mean that PB pretreatment influences ethylmorphine N-demethylation only quantitatively but not qualitatively. The experiments described here provide data which improve the characterization of cytochrome P-450. They are derived from the overall rate of N-demethylation reactions in microsomes, independently from conventional spectrophotometric methods.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00498783
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