Biomedical subjects
D M Ziegler
Publications and source records attributed to D M Ziegler.
Properties of an N,N-dimethyl-p-aminoazobenzene oxide reductase purified from rat liver cytosol.
Homogenates of all rat tissues examined, except brain, catalyze reduction of N,N-dimethyl-p-aminoazobenzene N-oxide (DMAB N-oxide) to N,N-dimethyl-p-aminoazobenzene by NADPH. Liver is the most active, and about one third of the homogenate activity of this tissue is recovered in the cytosol fraction. The purified cytosol enzyme has the properties of a tetrameric protein (Mr 370,000) consisting of identical subunits free from chromophores that absorb in the visible spectrum and from metals or other detectable prosthetic groups. The purified reductase is also free from NADPH oxidase and from cytochrome c or azo reductase activities. The enzyme is quite specific for NADPH as reductant and DMAB N-oxide as the electron acceptor. Reduction of other N,N-dimethyl-arylamine or alkylamine oxides as well as N-methylheterocyclicamine oxides could not be detected. Analysis of kinetic data indicate that, at saturating concentrations of the other substrate, 21 microM NADPH and 700 microM DMAB N-oxide are required for half maximal velocity. At infinite concentrations of both substrates the turnover is 150 min-1 at 37 degrees C.
Contribution of N-oxygenation to the metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by various liver preparations.
Liver microsomes from uninduced mice and rats catalyze NADPH- and oxygen-dependent N-oxygenation of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The N-oxide is the principal product and accounts for 95-96% of the total MPTP metabolized by microsomes. Demethylation of MPTP is detectable but the rate of nor-MPTP formation was never more than 4-6% of the rate of N-oxygenation. Studies on the biochemical mechanisms for N-oxygenation of MPTP suggest that this reaction is catalyzed exclusively by the flavin-containing monooxygenase. This conclusion is based on the effects of selective cytochrome P-450 inhibitors, positive effectors, and alternate substrates for the flavin-containing monooxygenase as well as on studies with the purified hog liver enzyme. MPTP is an excellent substrate for this monooxygenase with a Km of 30-33 microM. Limited studies with human liver whole homogenates suggest that N-oxygenation is also a major route for the metabolism of MPTP in man and the rate of N-oxide formation is approximately equal to the rate of mitochondrial monoamine oxidase-dependent MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium species) production.
Substrate specificity of the rabbit lung flavin-containing monooxygenase for amines: oxidation products of primary alkylamines.
Substrate activity of a flavin-containing monooxygenase isolated from rabbit lung microsomes has been examined with a number of primary, secondary, and tertiary amines. Of the secondary and tertiary amines tested, trifluoperazine, prochlorperazine, N, N-dimethyloctylamine, desmethylperazine, and N-methyloctylamine half-saturate the enzyme at concentrations less than 100 microM. Although the lung enzyme does not exhibit detectable substrate activity with primary arylamines, it catalyzes N-oxygenation of alkylamines to oximes. Studies on the mechanism for the oxidation of n-dodecylamine suggest that the amine is first oxidized to the hydroxylamine which is then further oxidized to the oxime. This interpretation is based on product identification, kinetic studies, and changes in the ratio of hydroxylamine to oxime formed as a function of initial substrate concentration. Kinetic constants calculated for the oxidation of n-dodecylamine and n-dodecylhydroxylamine indicate that the latter saturates the enzyme at a 100-fold lower concentration than that required for the parent amine, and the hydroxylamine is the dominant product only at saturating concentrations of the amine. The ratio of substrate-dependent NADPH and O2 consumption and product formation (hydroxylamine + 2 X oxime) is approximately 1.0:0.9:0.7. Although the reason for the less than stoichiometric yield of products is not known, uncoupling of the enzyme by primary amines does not appear to be a major factor since substrate-dependent increase in H2O2 formation is never more than 3% of substrate-dependent O2 consumption.
Studies on the biliary efflux of GSH from rat liver due to the metabolism of aminopyrine.
The biliary efflux of GSH and GSSG due to aminopyrine was studied using perfused rat livers. The infusion of 0.8 mM aminopyrine led to a rapid rise in the amount of GSH released into the bile with only a small increase in the amount of GSSG released; caval GSH + GSSG efflux was unaffected. N-Benzylimidazole, an inhibitor of cytochrome P-450, completely blocked the response while phenobarbital pretreatment of the rats doubled the rate of GSH efflux. H2O2 and selenium-containing glutathione peroxidase were not involved since livers from selenium-deficient rats perfused with aminopyrine released GSH at the same rate as control livers. Aminopyrine injected i.p. into conscious rats also stimulated biliary GSH efflux to the same extent as with perfused livers. Biliary release of GSH in the perfused livers could be duplicated by infusing formaldehyde. It is proposed that formaldehyde produced during the N-demethylation of aminopyrine by cytochrome P-450 combines reversibly with GSH to form S-hydroxymethylglutathione which is oxidized by formaldehyde dehydrogenase to S-formylglutathione. Formaldehyde formed in excess of its capacity to be metabolized enzymatically is released into the bile as S-hydroxymethylglutathione which then dissociates to its initial reactants.
Role of reversible oxidation-reduction of enzyme thiols-disulfides in metabolic regulation.
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Rabbit lung flavin-containing monooxygenase is immunochemically and catalytically distinct from the liver enzyme.
Flavin-containing monooxygenase has been purified to homogeneity from lung microsomes of pregnant rabbits. Antibody to rabbit lung flavin-containing monooxygenase was raised in guinea pig. Ouchterlony double diffusion analysis with this antibody produced precipitin lines of identity with the purified rabbit lung enzyme and lung microsomes from pregnant and non-pregnant rabbits. No cross-reaction was seen with liver microsomes from rabbit or with purified pig liver enzyme. The tricyclic antidepressant drugs, imipramine and chlorpromazine, are not substrates for the rabbit lung enzyme, whereas they are rapidly oxidized by the pig liver enzyme. These results indicate that rabbit lung and liver flavin-containing monooxygenases differ in substrate specificity, are immunochemically distinct proteins, and may be different gene products.
Increased biliary GSSG efflux from rat livers perfused with thiocarbamide substrates for the flavin-containing monooxygenase.
Thiourea, phenylthiourea, and methimazole perfused into rat liver stimulated the biliary efflux of GSSG without affecting the excretion of GSH into either the bile or the caval perfusate. The thiocarbamide moiety appears essential, since perfusion with urea, phenylurea, or N-methylimidazole did not stimulate GSSG release. Hydrogen peroxide is also not an obligatory intermediate, since thiocarbamide-induced GSSG efflux was undiminished in livers from selenium-deficient animals. The response was also not affected by N-benzylimidazole, a potent cytochrome P-450 inhibitor, which suggests that this monooxygenase is not involved. However, the results are consistent with a model based on S-oxygenation of thiocarbamides to formamadine sulfenates catalyzed exclusively by the flavin-containing monooxygenase. The resulting sulfenate is reduced by GSH, yielding GSSG and the parent thiocarbamide. Rapid cellular oxidation of GSH by this mechanism leads to biliary efflux of the disulfide.
Permeability of adolescent rat intestine to pancreatic ribonuclease.
There are indications that exocrine pancreatic enzymes may undergo an enteric recirculation. Ribonuclease (RNase) is present in considerable quantities in pancreatic exocrine secretions. Therefore we studied the intestinal transport of pancreatic RNase using everted gut sacs from duodenum, midgut, and ileum of young rats. Gut sacs were incubated aerobically at 37 degrees C, 22 degrees C, and 0 degrees C, and anaerobically at 37 degrees C. Measurable amounts of RNase crossed the intestinal barrier, but neither duodenum, midgut nor ileum absorbed RNase preferentially. The experimental data favor simple diffusion of RNase across rat small bowel. However, the amounts of pancreatic RNase absorbed were small. Thus, while our data do not negate the concept of an enteropancreatic recirculation in the rat, only a minor quantity of luminal, pancreatic RNase could recirculate.
Cytochrome P-450- and flavin-containing monooxygenase-catalyzed formation of the carcinogen N-hydroxy-2-aminofluorene and its covalent binding to nuclear DNA.
The metabolic N-oxidation of the carcinogen 2-aminofluorene was examined in vitro using fortified hepatic microsomes from a variety of species. Rat, dog, human, and pig liver microsomes catalyzed the formation of N-hydroxy-2-aminofluorene (N-OH-AF) from AF at rates of 1.6, 1.0, 1.2, and 3.5 nmol/min/mg protein, respectively. The involvement of both cytochrome P-450 and the flavin-containing monooxygenase was demonstrated with hepatic microsomes and with purified enzymes by using specific enzyme inhibitors. 2-[(2,4-Dichloro-6-phenyl)phenoxy]ethylamine, a potent cytochrome P-450 inhibitor, decreased microsomal N-OH-AF formation by 96, 83, 70, and 46% in the rat, dog, human, and pig, respectively; and further addition of methimazole, a high-affinity flavin-containing monooxygenase substrate, abolished the residual N-hydroxylating activity. Using the purified porcine flavin-containing monooxygenase, metabolic formation of N-OH-AF occurred at a rate of 4.9 nmol/min/nmol flavin adenine nucleotide and was insensitive to 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine inhibitor. In addition, purified rat liver cytochrome P-450 (isolated from 5,6-naphthoflavone-induced animals) N-hydroxylated AF (1.1 nmol/min/nmol P-450) and was completely inhibited by 2-[(2,4-dichloro-6-phenyl)-phenoxy]ethylamine, but the reaction was insensitive for methimazole. To determine whether or not the metabolic formation of N-OH-AF could lead directly to covalently bound adduct(s) with DNA under these incubation conditions (30 min, pH 7.5), the binding of synthetic and metabolically formed [3H]-N-OH-AF to added calf thymus DNA and to DNA in isolated rat liver nuclei was investigated. In all cases, the amount of DNA-bound carcinogen accounted for 0.08 to 0.15% of the N-OH-AF present in the incubation mixtures. These data, when compared to the levels of AF bound to hepatic nuclear DNA reported in vivo, suggest that the nonenzymatic reaction of N-OH-AF with nuclear DNA may be sufficient to account for a substantial portion of the observed in vivo binding of this carcinogen.
Changes in dimethylaniline N-oxidase activity of mouse liver and kidney induced by steroid sex hormones.
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Hamster hepatic nuclear mixed-function amine oxidase: location and specific activity.
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The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies.
A FAD-containing monooxygenase isolated from pig liver microsomes migrates as a single band upon electrophoresis in polyacrylamide gels in the presence of dodecyl sulfate. The minimum molecular weight based on mass of amino acids per mole of flavin is 64,000. However, the catalytically active enzyme exists as aggregating units of the monomer. Neither oxygen nor organic substrates perturbed the spectrum of the oxidized flavoprotein and their binding to this form of the enzyme could not be detected. Anaerobically NADPH bleaches the flavoprotein, and in the presence of both NADPH and oxygen a remarkably stable intermediate form of the enzyme, with an absorption band at 375 nm, is observed. The spectrum of the intermediate resembles that of a peroxyflavin. The monooxygenase catalyzes NADPH- and oxygen-dependent oxygenations of nucleophilic nitrogen- or sulfur-containing compounds. Kinetic studies carried out with a model organic nitrogen substrate (trimethylamine) and a sulfur substrate (methimazole) gave similar patterns. The kinetic data are consistent with an ordered Ter-Bi mechanism with an irreversible step between the second and third substrate where NADPH is added first, followed by oxygen, and the oxidizable organic substrate is added last. If NADPH is the first substrate added, then NADP+ must be the last product released since NADP+ is competitive with NADPH.
Studies on the nature and regulation of the cellular thio:disulphide potential.
Microsomal fractions separated from homogenates of liver, kidney and corpora lutea contain a monooxygenase (dimethylaniline monooxygenase [N-oxide forming], EC 1.14.13.8) that catalyses NADPH- and oxygen-dependent oxidation of cysteamine to cystamine. The monooxygenase purified to homogeneity from hog liver also catalyses oxygenations of diverse xenobiotics, but it does not catalyse oxidation of any other physiological sulphur- or nitrogen-containing compounds. All the available evidence indicates that cysteamine is the physiological substrate for the monooxygenase, and the oxidation of this thiol to the disulphide may be a significant source of disulphide maintaining the cellular thiol:disulphide potential. The concentration of protein-low molecular weight mixed disulphide is a function of this potential. Changes in concentration of this protein-mixed disulphide reflect changes in thiol:disulphide balance. At constant substrate concentrations the potential would depend primarily on activity of the cytosol glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) relative to that of the membrane-bound monooxygenase. In hepatic tissue from adult mice and hamsters there is a correlation between the concentration of protein-mixed disulphide and the activity of the monooxygenase relative to the reductase. Hepatic glutathione reductase is relatively constant in mice, but the monooxygenase is much higher in the female than in the male. After gonadectomy monooxygenase activity decreases in the female and increases in the male. Activities are restored to control levels by treating males with testosterone and females with progesterone. Testosterone decreases and progesterone increases activity. These two hormones apparently regulate the level of this enzyme in hepatic tissue.
S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase.
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Hepatic microsomal mixed-function amine oxidase.
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Intermediate metabolites of thiocarbamides, thioureylenes and thioamides: mechanism of formation and reactivity.
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The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions.
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