Interaction of Mixed Function Oxidase with its Substrates and Associated Redox Transitions of Cytochrome P‐450 and Pyridine Nucleotides in Perfused Rat Liver

Abstract

1 The interactions of a ‘Type I' compound, hexobarbital, and of a ‘Type II’ compound, metyrapone, with the mixed function oxidase were demonstrated by spectrophotometric techniques in hemoglobin-free perfused livers from phenobarbital-treated rats. The binding signal for hexobarbital occurred when cytochrome P-450 was mainly oxidized (normoxic perfusion) or reduced (anoxic perfusion). The binding signal disappeared as hexobarbital was metabolized. 2 Functional aspects were demonstrated by simultaneous readings of (a) the reduced cytochrome P-450-CO complex, providing an indication of the degree of reduction of cytochrome P-450; (b) surface fluorescence (366 → > 420 nm); and (c) oxygen uptake. The steady state level of reduced P-450-CO was increased when substrate was metabolized. Surface fluorescence intensity decreased following hexobarbital addition. This decrease was explained partly by a decrease of the tissue level of NADPH, and partly also by interference of the P-450-hexobarbital complex at the excitation wavelength. Extra oxygen uptake was 2.2–2.4 moles per mole of hexobarbital. Half-maximal concentration of hexobarbital was 59 and 75 μM for extra oxygen uptake and binding to the oxidase, respectively, in agreement with data for enzymatic activity and binding from isolated liver microsomes. The parameters (a, b, c) returned to their original level as hexobarbital was metabolized, similar to findings with the steroid 21-hydroxylase system of isolated adrenocortical microsomes. 3 The overall degree of reduction of the NADPH system, NADPH/NADPH + NADP+, decreased from 0.8 to 0.7 in livers from phenobarbital-treated rats when hexobarbital was added. In contrast, the free cytosolic NADH system did not undergo redox changes as indicated by a constant ratio of lactate/pyruvate in the perfusate. 4 Mitochondrial NADH dehydrogenase was shielded against the barbiturate by the endoplasmic reticulum as a consequence of phenobarbital induction.