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D M Ziegler

Publications and source records attributed to D M Ziegler.

At least 19 recordsLinked to original sources
PubMed1992

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.

Animals↗
PubMed1991

Cloning, primary sequence, and chromosomal mapping of a human flavin-containing monooxygenase (FMO1).

cDNA clones that code for a pig and human flavin-containing monooxygenase (FMO) have been isolated. The full-length sequence of the human cDNAs revealed that they encode a polypeptide of 532 amino acid residues containing putative FAD- and NADP-binding sites. The deduced amino acid sequence has 88 and 86% identity, respectively, with the pig and rabbit "hepatic" forms of FMO, but is only 58% similar to the rabbit "pulmonary" FMO, and thus represents the human ortholog of the "hepatic" form of FMO. However, as this FMO is present in low abundance in human adult liver, the general term "hepatic" for this form of the enzyme is misleading, and thus we propose the name FMO1 to describe this human FMO and its mammalian orthologs. Northern blot analysis demonstrated that human FMO1 mRNA is more abundant in fetal than in adult liver, indicating that in man the enzyme is subject to developmental regulation. Southern blot hybridization of human genomic DNA suggests that the protein is encoded by a single gene, which has been designated FMO1 and mapped to chromosome 1.

Amino Acid Sequence↗
PubMed1991

Estimation of flavin-containing monooxygenase activities in crude tissue preparations by thiourea-dependent oxidation of thiocholine.

The activity of flavin-containing monooxygenases in microsomes and whole homogenates is readily estimated by following the thiourea-dependent oxidation of thiocholine. NADPH- and oxygen-dependent flavin-containing monooxygenases catalyze the oxidation of thiourea to formamidine sulfenic acid, which oxidizes thiocholine to thiocholine disulfide. The latter reaction is quite rapid and never rate limiting even at concentrations of thiocholine below 30 microM. The loss of thiocholine in deproteinized aliquots of the reaction medium is measured colorimetrically with the thiol reagent, DTNB [5,5'-dithiobis(2-nitrobenzoate)]. In the absence of thiourea, thiocholine is not oxidized and its disulfide is not reduced at a detectable rate even in reactions containing 4-5 mg of liver or kidney homogenate protein per milliliter. In all tissues where both can be measured, rates of thiocholine oxidation and N,N-dimethylaniline N-oxygenation were virtually identical, which suggests that both activities are catalyzed by the same monooxygenase.

Aniline Compounds↗
PubMed1991

Effects of dietary protein-energy interrelationships on Holstein steer performance and ruminal bacterial fermentation in continuous culture.

In vivo and in vitro 3 x 2 factorial experiments were conducted concurrently to evaluate the incorporation of 0, 15, or 30% sugar beet pulp (SBP) as an energy source in diets fed to growing Holstein steers with either soybean meal (SBM) or alcohol-treated, defatted soybean flakes (ATSBF) as primary supplemental protein sources. Three groups of 42 Holstein steers each were fed six different diets from 54 kg initial BW to 320 kg in three experimental periods. There were no overall SBP level x protein source interactions (P greater than .05). Beet pulp level tended to decrease ADG (linear, P = .05) and increase feed/gain (linear, P less than .05) and DMI (quadratic, P less than .05). Each grower diet was used in a substrate for ruminal microbial metabolism in six dual-flow, continuous-culture fermenters. Organic matter and carbohydrate digestion were similar (P greater than .05) among diets. Increasing dietary levels of SBP caused a concomitant increase (P less than .05) in acetate and decrease (P less than .05) in butyrate and isobutyrate concentrations. Beet pulp level x protein source interactions (P less than .05) were observed for CP degradation, ammonia and nonammonia N, and dietary N flow. Crude protein degradation was higher (P less than .05) for the 0% SBP with SBM diet (81.3%) than for the 30% SBP with ATSBF diet (64.4%). Efficiency of bacterial synthesis was similar (P greater than .05) among diets. Results indicated that SBP is an effective dietary energy source for high-energy grower diets at 15 or 30% of the DM but may cause a decrease in some performance traits. There were no nutritional benefits of using ATSBF vs SBM as the supplemental N source.

Animal Feed↗
PubMed1990

The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene.

The primary sequence of the flavin-containing monooxygenase expressed in pig liver has been derived from the nucleotide sequence of cloned cDNA. The derived sequence is composed of 532 amino acids and represents a protein having a molecular weight of 58,952. The complete sequence was obtained from a single clone containing 2070 bases. A second clone, obtained from an independent library, yielded an identical sequence for the 1374 bases present. The amino acid composition compiled from the derived sequence is very similar to that obtained previously from the purified protein. In addition, a 10 amino acid sequence in a peptide formed from the purified protein by digestion with V8 protease exactly matches the derived sequence for residues 309-318. The flavin-containing monooxygenase expressed in pig liver is also expressed in pig lung and kidney as determined by analysis of both microsomal proteins and mRNA. The ratio of mRNA to protein for the enzyme in kidney is about 5 times greater than the same ratio for liver and about twice the ratio for lung. The reasons for these differences are not understood. Southern analysis of genomic DNA indicates that there is a single gene encoding the flavin-containing monooxygenase expressed in pig liver. Therefore, the broad activity of this enzyme in liver appears to be the result of the catalytic diversity of a single protein.

Amino Acid Sequence↗
PubMed1990

Flavin-containing monooxygenases: enzymes adapted for multisubstrate specificity.

Unlike all other oxidases, microsomal flavin-containing monooxygenases (FMO) discriminate between essential and foreign compounds by excluding the former rather than selectively binding the latter. As Daniel Ziegler describes here, xenobiotics that readily cross cell membranes can enter the catalytic cavity, whereas charged groups on essential metabolites that prevent their passive diffusion out of the cell also block their access to FMO. FMO appears to be ideally adapted to catalyse the detoxification of structurally diverse soft nucleophiles (e.g. alkaloids with basic side-chains and organic sulfur xenobiotics) so abundant in food derived from plants.

Animals↗
PubMed1990

Evaluation of various nitrogen supplements in starter diets for growing Holstein steers and their effects on ruminal bacterial fermentation in continuous culture.

Concurrent in vivo and in vitro studies were conducted to evaluate urea (U), soybean meal (SBM), ground soybeans (RAW), extruded soybeans (ES) or extruded soybeans plus urea (ES + U) as primary supplemental N sources in starter diets for Holstein steers. Three groups of 48 Holstein steers each were fed five different starter diets to 181 kg BW in three experimental periods over 2 yr. Average daily gains were similar (P greater than .05) for steers fed ES + U (1.12 kg), ES (1.08 kg) and SBM (1.09 kg) but lower (P less than .05) for those fed U (1.00 kg) or RAW (.97 kg) diets. Feed/gain was similar (P greater than .05) for ES-fed steers vs those fed other diets except U. From 181 to 477 kg, all steers were fed the same diet. Steers fed the RAW starter diet had the lowest (P less than .05) ADG for the entire period. The starter diets were used as substrates for ruminal microbial metabolism in eight dual-flow continuous culture fermenters. True OM digestion was higher and NDF and ADF digestion was lower (P less than .05) for the ES + U diet than for the ES diet. Dietary protein degradation was lowest (P less than .05) for the ES diet (64.4%). Total bacterial N flow was higher (P less than .05) with the ES + U, SBM and U diets than with the ES diet. Lysine flow was higher (P less than .05) for the ES + U diet than for all other diets except ES. Results of these experiments indicate that ES as a protected ruminal escape N source with or without added urea did not improve steer performance above that obtained from SBM in starter diets.

Animal Feed↗
PubMed1979

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.

Amino Acids↗
PubMed1979

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.

Animals↗