Influence of N, N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c( P-450) reductase in a reconstituted rabbit liver microsomal enzyme system

Abstract

N, N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c( P-450) reductase (NADPH: ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 ( P-450LM 2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve ( n H = 1.7) is transformed to a straight line ( n H = 1.01) when the N-terminal reductase-binding domain in the P-450LM 2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N, N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N, N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM 2.