Search PubMed⌕ Search

Biomedical subjects

D M Ziegler

Publications and source records attributed to D M Ziegler.

88 records · Page 5Linked to original sources
PubMedDate not supplied

Substrate specificities of rabbit lung and porcine liver flavin-containing monooxygenases: differences due to substrate size.

Phenothiazine, 2-(trifluoromethyl)phenothiazine, and a series of 10-(N,N-dimethylamino-alkyl)-2-(trifluoromethyl)phenothiazines with alkyl side chains varying in length from C2 to C7 were tested for substrate activity with purified rabbit lung and porcine liver flavin-containing monooxygenases (FMO). While all were substrates for the hepatic FMO, only phenothiazines bearing C6 and C7 alkyl side chains were oxidized at significant rates by the pulmonary FMO. Kinetic constants calculated from reaction velocities for the oxidation of thiourea, phenylthiourea, and naphthylthiourea indicate that a nucleophilic heteroatom on the end of a molecule not much larger than a six-membered ring in cross section is oxidized by both enzymes, but the addition of bulky lipophilic substituents increases the Km of N-substituted thioureas for rabbit lung FMO and 1,3-diphenylthiourea (thiocarbanilide) is excluded entirely. From the dimensions of compounds excluded and from those oxidized, it would appear that the hydroperoxyflavin in rabbit lung FMO lies about 6-8 A below the surface in a channel no more than 8 A in diameter in its longest axis. The channel leading to this oxidant in hepatic FMO appears more open and readily admits compounds bearing a tricyclic ring. Differences in dimensions of the substrate channel appear responsible for some of the differences in substrate specificities between liver and lung FMO.

Animals↗
PubMedDate not supplied

NADPH-dependent oxidation of reduced ebselen, 2-selenylbenzanilide, and of 2-(methylseleno)benzanilide catalyzed by pig liver flavin-containing monooxygenase.

The selenazole ring-opened metabolites of ebselen, 2-selenylbenzanilide and 2-(methylseleno)benzanilide, are substrates for flavin-containing monooxygenase from pig liver. The Km values were 25 and 3 microM, respectively, measured at 37 degrees C, pH 7.4, in the presence of 1 mM GSH. The Vmax values were 390 mU/mg of protein, similar to those obtained with methimazole or other substrates for FMO1. Although ebselen also appears to be a substrate in the absence of GSH, it progressively inactivates the enzyme, apparently by binding covalently to essential enzyme thiols. The oxidation products of the selenol and methylseleno derivatives are rapidly reduced by GSH, regenerating the parent substrates. Rapid reduction of the selenide oxide by GSH was unexpected and suggests that, unlike S-oxidation of sulfides, Se-oxidation of selenides may be a route for bioactivation. The data show that in the presence of FMO1 micromolar amounts of either of these ring-opened metabolites establish a futile cycle catalyzing the oxidation of GSH to GSSG by NADPH and oxygen.

Anilides↗
PubMedDate not supplied

Inhibition of purified and membrane-bound flavin-containing monooxygenase 1 by (N,N-dimethylamino)stilbene carboxylates.

(E)-[2-(4-(Dimethylamino)phenyl)vinyl]benzenes bearing a nitrile or carboxyl group in the 2', 3', or 4' position were synthesized and tested for substrate activity with purified pig liver flavin-containing monooxygenase (FMO1). Although the nitrile derivatives were too insoluble to saturate the catalytic site at pH 7.4, they appeared to be substrates with K(m)'s somewhat above their maximum solubility (approximately equal to 0.1 mM) in the assay medium. Of the three carboxylic acid analogs, (E)-4-[2-(4(dimethylamino)phenyl)vinyl]benzoic acid had no detectable water solubility at pH 7.4, and measurements were restricted to (E)-3-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS3CO) and (E)-2-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS2CO). While DS3CO and DS2CO were substrates, they also inhibited FMO1 turnover. DS3CO was the more effective inhibitor, and at 2 mM it inhibited FMO1 and microsomal-catalyzed oxidation of methimazole (N-methyl-2-mercaptoimidazole) by 80-90%. Kinetic studies indicated that the aminostilbene carboxylates were noncompetitive with both the xenobiotic substrate, methimazole, and NADPH. However, inhibition constants calculated from double reciprocal plots of velocity vs NADPH were K(i)(comp) 130 and 150 microM for DS3CO and DS2CO, respectively, whereas the uncompetitive Ki's were 10-15 times higher, which suggests that inhibition of NADPH binding may be primarily responsible for inhibition of FMO1 by the aminostilbene carboxylates. This model is also consistent with inhibition of cyclohexanone monooxygenase, a bacterial analog of FMO. DS3CO and DS2CO were again noncompetitive with methimazole but primarily competitive with NADPH. The aminostilbene carboxylates had no detectable effects on activity of pig or rat liver NADPH-cytochrome P450 reductase, which suggests that they are not nonspecific flavoprotein antagonists.

Animals↗
PubMedDate not supplied

Oxidation of aldose reductase inhibitors ALO-4114 and ALO-3152 catalyzed by liver microsomes.

Rat and Cynomolgus monkey liver microsomes catalyze the oxidation of 2.7-difluoro-4.5-dimethoxyspiro (9H-fluorene-9,4'-imidazolidine)-2',5'-dione (ALO-4114) to its monomethoxymetabolite (ALO-4417). Formation of this product by O-demethylation of ALO-4114 is catalyzed by NADPH and oxygen-dependent microsomal enzymes with the properties of P-450 monooxygenases. The reaction is blocked by inhibitors selective for these enzymes and activity increases about 2-fold in rats pretreated with phenobarbital or methylcholanthrene. The increase in the O-demethylation of ALO-4114 was, however, considerably less than the increase in benzphetamine N-demethylation or nitrophenetole O-deethylation activities in liver microsomes from rats pretreated with either phenobarbital or methylcholanthrene. Rats pretreated with 20 or 40 mg/kg of ALO-4114 for 3-4 days failed to change significantly the rate of ALO-4114 O-demethylase activity of liver microsomes. O-Demethylation of the achiral ALO-4114 yields the chiral ALO-4417. The enantiomers separated on a Daicel Chiracel AS column by HPLC indicated that O-demethylation of ALO-4114 by microsomes from untreated rats was only slightly stereoselective. However, rats pretreated with methylcholanthrene not only enhanced activity, but also increased the formation of one enantiomer. Further oxidative metabolism of the enantiomers was slow and barely detectable in vitro. Studies conducted with Cynomolgus monkey liver microsomes from one male and one female per experimental group were generally consistent with those from the rat, but some differences were noted. Whether the differences are real or only reflect individual variations caused by the small sample size is not known at present.

Aldehyde Reductase↗
PubMedDate not supplied

The role of flavin-containing monooxygenase in the N-oxidation of the pyrrolizidine alkaloid senecionine.

The pyrrolizidine alkaloid, senecionine, is N-oxidized by purified pig liver flavin-containing monooxygenase but not by purified rabbit lung flavin-containing monooxygenase. The activity of the pig liver enzyme toward senecionine was linear with time and amount of enzyme. The oxygenation was not due to some indirect mechanism, such as O2- release from the enzyme, as scavengers of activated oxygen had no effect on product formation. The Km of purified pig liver flavin-containing monooxygenase for senecionine was 0.3 mM. Although senecionine is a substrate for the pig liver enzyme, studies performed with rat liver microsomes suggest that, in this species, cytochromes P-450 catalyze the majority of senecionine-N-oxidation. These experiments included inhibition by chemical inhibitors of P-450, treatment of the microsomes with elevated temperatures, inhibition by anti-NADPH-cytochrome P-450 reductase antibody, the effect of dexamethasone on N-oxidation, and relative amounts of flavin-containing monooxygenase determined by immunoquantitation. These results demonstrate that flavin-containing monooxygenase can be involved in the detoxication of pyrrolizidine alkaloids via N-oxidation, but the relative contribution of flavin-containing monooxygenase and cytochromes P-450 may be species and tissue dependent.

Animals↗
PubMedDate not supplied

Microsomal mixed-function amine oxidase. Oxidation products of piperazine-substituted phenothiazine drugs.

Oxidation products of fluphenazide, thioproperazine, and trifluoperazine obtained in reactions catalyzed by homogeneous preparations of the microsomal mixed-function amine oxidase have been isolated and identified. Approximately 0.5 g of metabolite of each piperazine-substituted phenothiazine drug was prepared in reactors containing, as catalyst, the purified oxidase covalently attached to glass beads. Nuclear magnetic resonance spectra of the isolated products indicated that with all three substrates the enzyme preferentially catalyzes N-oxidation of the piperazine nitrogen furthest from the phenothiazine nitrogen atom. The enzyme-catalyzed oxidation is quite specific and oxidation of the sulfur or nitrogen atoms in the phenothiazine ring could not be detected. Concentrations of piperazine-substituted phenothiazines required to half-saturate the amine oxidase were in the micromolar range and at pH 8.3 and 37 degrees C, all those tested were oxidized at approximately 2 mumol/min/mg of enzyme. Kinetic constants for the piperazine-substituted phenothiazines were very similar to those obtained with phenothiazines containing a dimethylaminopropyl sidechain.

Animals↗
PubMedDate not supplied

Oxidation of desmethylpromethazine catalyzed by pig liver flavin-containing monooxygenase. Number and nature of metabolites.

Homogenous preparations of pig liver flavin-containing monooxygenase catalyze the oxidation of desmethylpromethazine to six distinct metabolites. The products, identified with the aid of chemically synthesized reference compounds, arise by N-oxygenation of the side-chain nitrogen and by S-oxygenation of the phenothiazine ring sulfur. Although kinetic constants (KM) differ somewhat for the R and S isomers, both are initially oxidized to either the desmethylpromethazine sulfoxide or a secondary hydroxylamine. All other metabolites detected are formed by further oxidation of the latter product. Reaction rates determined with intermediates indicate that the secondary hydroxylamine is oxidized to an oxime sulfoxide via the following intermediates: nitrone-->N-hydroxydidesmethylpromethazine-->oxime-->oxime sulfoxide. All of the steps, except for the hydrolysis of the nitrone, are enzymic, and the data presented demonstrate unambiguously for the first time that the oxidative steps in the metabolism of a secondary amine to the oxime are enzymic. In addition, sulfoxidation of desmethylpromethazine is the first demonstration that flavin-containing monooxygenase can catalyze sulfoxidation of a phenothiazine drug bearing a basic side-chain nitrogen.

Amines↗