Abstract
Cytochrome P-450 isozyme 3a, isolated from hepatic microsomes of rabbits treated chronically with ethanol, was found to have a unique substrate specificity when compared with isozymes 2, 3b, 3c, and 4. Form 3a has unusually high activity in the p-hydroxylation of aniline and in the oxidation of alcohols to aldehydes. These properties are reflected in the increased activities of these substrates in microsomes from ethanol-treated rabbits as compared to microsomes from untreated animals or those administered phenobarbital or 5,6-benzoflavone. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH cytochrome P-450 reductase and is stimulated by the presence of phospholipid, was shown not to be due to contaminating catalase or an NAD- or NADP-dependent alcohol dehydrogenase. Isozyme 3a catalyzes the oxidation of methanol, 1-propanol, and 1-butanol as well as ethanol; the relationships between the apparent Km values for these alcohols and their octanol-water partition coefficients is in accord with the known hydrophobic nature of the P-450 binding site. Whereas typical substrates of isozyme 2 are known to be metabolized with the stoichiometry predicted of a monooxygenase reaction, with isozyme 3a the sum of acetaldehyde formed from ethanol and of hydrogen peroxide generated is inadequate to account for the NADPH and oxygen consumed. Free hydroxyl radicals appear to mediate the slow oxidation of ethanol in the presence of the reductase alone but not the faster rate catalyzed by P-450 isozyme 3a. The results obtained, however, do not rule out the involvement of hydroxyl radical equivalent generated and bound at the active site of the cytochrome.
Highlights
Cytochrome P-460 isozyme 3a,isolated from hepatic of P-450’) was not seen with other known inducers [8, 9], it microsomes of rabbits treatedchronically with ethanol, has been postulated that ethanol induces a P-450 isozyme was found to have a unique substrate specificity when which is distinct[8, 9, 11]from the known inducible forms.In compared with isozymes 2, 3b, 3c, and 4
Form 3a has support of this idea, spectral binding studies and inhibition unusually high activity in the p-hydroxylation of ani- experiments have demonstrated increased affinity of rat heline and in theoxidation of alcoholsto aldehydes. These patic microsomal P-450 forcyanide, tetrahydrofuran,and properties are reflected in the increased activities of Me2S0(9, 11, 13, 14) after chronic ethanol treatment
The existence of anNADPH-dependent,catalase-and ADH-independent “microsomaelthanol-oxidizing system” was first proposed by Lieber and DeCarli [15]. Such activity was reported to be increased by chronic ethanol administration [12, 15,16,17], but the abilityof microsomal cytochrome P450 to catalyze the oxidation of ethanol and other aliphatic alcohols [18, 19] and the possible physiological significance of ent alcohol dehydrogenase
Summary
The existence of anNADPH-dependent,catalase-and ADH-independent “microsomaelthanol-oxidizing system” was first proposed by Lieber and DeCarli [15] Such activity was reported to be increased by chronic ethanol administration [12, 15,16,17], but the abilityof microsomal cytochrome P450 to catalyze the oxidation of ethanol and other aliphatic alcohols [18, 19] and the possible physiological significance of ent alcohol dehydrogenase. In the present paperwe report the effects of chronic ethanol administration anodf other inducers on hepatic microsomal activities and compare the catalytic properties of form 3a withthose of other purified P-450 isozymes from rabbit liver in the reconstituted nonliposomal. The purification of P - 4 5 0 ~ ~ .f,r,om liver microsomes of ethanol-treated rabbits has recently been described [28]; the preparations had specific contents of 16 to 20.
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