Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.
The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).
Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats. The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).