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L L Poulsen

Publications and source records attributed to L L Poulsen.

At least 19 recordsLinked to original sources
PubMed2000

Redox regulation of yeast flavin-containing monooxygenase.

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.

Base Sequence↗
PubMed1999

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae.

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.

Amino Acid Sequence↗
PubMed1999

Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum.

The flavin-containing monooxygenase from yeast (yFMO) catalyzes the O2- and NADPH-dependent oxidations of biological thiols, including oxidation of glutathione to glutathione disulfide (GSSG). Glutathione and GSSG form the principle redox buffering system in the cell, with the endoplasmic reticulum (ER) being more oxidizing than the cytoplasm. Proper folding of disulfide-bonded proteins in the ER depends on an optimum redox buffer ratio. Here we show that yFMO is localized to the cytoplasmic side of the ER membrane. We used a gene knockout strain and expression vectors to show that yFMO has a major effect on the generation of GSSG transported into the ER. The enzyme is required for the proper folding, in the ER, of test proteins with disulfide bonds, whereas those without disulfide bonds are properly folded independently of yFMO in the ER or in the cytoplasm.

Cell Compartmentation↗
PubMed1996

Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae.

An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors. A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and characterized. The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity. The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively. The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine. This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals.

Cloning, Molecular↗
PubMed1995

Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity.

Kinetic studies on mechanism of the flavin-containing monooxygenase (FMO1) from pig liver microsomes are described in detail with special emphasis on the interpretation of constants derived from the rate equation. The evidence reviewed indicates that oxidation of xenobiotic substrates by the 4a-hydroperoxyflavin form of the enzyme is a second order reaction not saturable by substrate. Under steady-state conditions decomposition of the hydroxyflavin (an intermediate form of the enzyme that does not require enzyme-substrate or enzyme-product equilibrium complexes) is rate limiting. The lack of detectable equilibrium binding is also consistent with rate constants defining Km deduced from steady-state measurements. A model consistent with all evidence currently available indicates that at saturating concentrations of xenobiotic substrates that catalytic site on the enzyme is unoccupied most of the time. This property may explain why non-substrate analogs of xenobiotic substrates do not inhibit FMO activity. Rate constants for the oxidation of xenobiotics by the enzyme-bound and synthetic 4a-hydroperoxyflavin indicate that while enzyme protein accelerates the reaction with xenobiotics bearing nitrogen, it has only marginal effects on the oxidation of substrates bearing sulfur. Differences in the nucleophilicity of compounds bearing these heteroatoms may be primarily responsible but other, as yet undefined, factors may also contribute. In addition, analysis of rate constants affected by protonated lipophilic amines indicates that these allosteric effectors apparently modify enzyme structure so as to affect two or more rate constants and, depending on the nature and concentration of the xenobiotic substrate, protonated amines can either stimulate or inhibit catalytic activity.

Amines↗
PubMed1995

Oxidation of aldehydes catalyzed by pig liver flavin-containing monooxygenase.

Flavin-containing monooxygenase-1 (FMO1) purified to homogeneity from pig liver microsomes catalyzes NADPH- and oxygen-dependent oxidation of salicylaldehyde to pyrocatechol and formate. These products, formed in equimolar amounts, were the only ones detected that suggests that FMO1 catalyzes the oxidation of salicylaldehyde by Baeyer-Villiger chemistry. In addition to salicylaldehyde, 2-hydroxy-1-naphthaldehyde, 5-chlorosalicylaldehyde, 5-nitrosalicylaldehyde, ferrocene carboxaldehyde, 2-pyridine carboxaldehyde, and acetylacetone also stimulated NADPH-dependent oxygen uptake in the presence of FMO1. On the other hand, benzaldehyde, 2-methoxybenzaldehyde, 4-pyridine carboxaldehyde and 3- or 4-hydroxybenzaldehyde, and none of the alkylaldehydes tested had detectable substrate activity. Pig liver FMO1 apparently only catalyzes C-oxidation of reactive aldehydes with a hydrogen ion acceptor function adjacent to the carbonyl.

Aldehydes↗
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↗
PubMed1988

Estimation of lipid concentrations on thin-layer plates by densitometry of transparent copies.

A relatively simple procedure for the quantitative estimation of phospholipids resolved on thin-layer plates has been developed. After resolution in an appropriate solvent, the lipids are visualized by staining with iodine, ninhydrin, or molybdate and then photocopied onto transparent sheets with a standard office copy machine. The density of each spot on the photocopy, measured with a simple silicon cell area densitometer, is a direct function of each lipid applied to the plate over at least a six- to eightfold range in concentration. Under controlled conditions the staining and photocopying steps are quite reproducible. Known concentrations of the choline, ethanolamine, serine, and inositol derivatives of L-alpha-phosphatidic acid applied either separately or as mixtures can be determined essentially quantitatively (100 +/- 5%) by this procedure following their resolution on the thin-layer plates.

Animals↗
PubMed1986

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.

Amines↗
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↗
PubMed1977

Purification and crystallization of NADP+-specific isocitrate dehydrogenase from Escherichia coli using polyethylene glycol.

A simple and rapid method is presented for purifying the NADP+-dependent isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), from Escherichia coli, which relies on fractionation of the enzyme with polyethylene glycol. The shortened preparation results in a 32% relative recovery of purified enzyme at a specific activity of 127 micronmol/min per mg of protein. The Km values for threo-DS-isocitrate, NADP+, NAD+, Mg2+ and Mn2+ are 6.4, 36, 3000, 19.7 and 2.0 micronM, respectively. The stability of the enzyme as a function of dilution and temperature are also reported. Recrystallization of the purified enzyme under different conditions readily produces a variety of single crystals. Crystals grown from ammonium sulfate solutions belong to monoclinic space group C2 with a = 125 A, b = 111 A, c = 83.5 A and beta = 108degrees 45'. Density measurements of these crystals indicate there are two 80 000-dalton dimers per asymmetric unit.

Crystallization↗