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 11' 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 --f >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 a t 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 pM 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. I n contrast, the free cytosolic NADH system did not undergo redox changes as indicated by a constant ratio of lactatelpyruvate in the perfusate.4. Mitochondria1 NADH dehydrogenase was shielded against the barbiturate by the endoplasmic reticulum as a consequence of phenobarbital induction.A wealth of information about the hepatic mixed function oxidase system has accumulated during recent years from studies with isolated microsomal fractions [1, 1 a]. Direct insight into this system within the intact organ is possible by spectrophotometric and fluorometric techniques in the hemoglobin-free perfused rat liver [2,3]. I n the present study, simultaneous measurement of the effect of external substrate on four parameters is described, furthering the understanding of this system when , integrated in the dynamics of the cell: (a) spectral interaction of external substrate with cytochrome P-450, the terminal oxidase ; secondly, the steady state level of the reduced P-450-CO complex, both Enzyme. Mixed function oxidase (EC 1.99.1.1.). 35.measured by spectrophotometry of perfused liver in the transmitted light; (b) effect of external substrate on reduced pyridine nucleotide surface fluorescence; and (c) oxygen uptake, serving as a measure of the rate of mixed function oxidation.The interrelations between mixed function oxidase and other cellular redox systems such as the ...

State of Oxidation-Reduction and State of Binding in the Cytosolic NADH-System as Disclosed by Equilibration with Extracellular Lactate/Pyruvate in Hemoglobin-Free Perfused Rat Liver

“…Similar methods might be applied for analysis of contributions of other systems. For example, specific transitions in extramitochondrial [27,28] Fig. 11) and acetate (Fig.…”

The steady state level of catalase compound I in isolated hemoglobin‐free perfused rat liver

“…Dual wavelength absorbance photometry by sinusoidal wavelength modulation [7] was used for more sensitive study of absorbance changes as a function of time (see also [8]). Bandwidth of monochromatic light was 4 nm.…”

Stimulation of mixed‐function oxidation of 7‐ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol

“…Since the P-450 -CO complex increased upon the addition of hexobarbital in livers from fed, phenobarbital-treated rats, Sies a n d Brauser concluded that NADPH supply was not ratelimiting in livers from fed, phenobarbital-treated rats [27]. It would be interesting, therefore, t o identify factors responsible for the metabolism of drugs other than 7-ethoxycoumarin in periportal and pericentral regions of the liver in various nutritional states.…”