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[Study of the relationship between analgesic activity and structure of synthetic melanocortin analogs].

Melanocortins, peptides of the family of adrenocorticotropic (ACTH) and melanocyte-stimulating (MSH) hormones, have a wide range of physiological activity. Heptapeptide semax (MEHFPGP) is an analog of the ACTH4-10 fragment. Previously, pronounced nootropic and neuroprotective activities were shown for this peptide; in addition, it decreases pain sensitivity in animals. In this work, the relationship between analgesic activity of the peptide and its structure was studied. The following analogs of the N-terminal ACTH fragments were used: rMEHFPGP, where r is glucuronic acid, KEHFPGP, GEHFPGP, EHFPGP, HFPGP, and ERP. The peptides were administered intraperitoneally at doses of 0.015, 0.05 and 0.5 mg/kg. Rat pain sensitivity was assessed using the paw-withdrawal test. Truncations of the N-terminal residues eliminated the analgesic activity. The peptide analgesic effect was observed after the replacement of the N-terminal methionine with lysine or after the attachment of glucuronic acid to the methionine, while the peptide with glycine instead of the methionine had no effect on pain sensitivity. Hence, the amino acid at position 1 of semax analogs plays a key role in mediating the analgesic effects of the peptides.

Adrenocorticotropic Hormone↗
PubMed1987

Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea.

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecular-mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the O-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.

Animals↗
PubMed1994

Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus epidermidis.

Slime-producing coagulase-negative staphylococci have emerged as important pathogens especially in immunocompromised hosts and patients with implanted devices. Although the extracellular slime layer is considered an important virulence factor, the chemical composition of the slime polysaccharide(s) remains unknown. The crude slime product derived from two reference Staphylococcus epidermidis strains (ATCC 35983 and 35984) and two clinical isolates was found to contain protein (11-20.5%), hexosamines (8-19%), neutral sugars (12.2-14%), phosphates (4-9.5%), uronic acids (1-13%), and small amounts of sulfates (0.5-3%). Preparative anion-exchange chromatography separated a main carbohydrate component which was isolated by combined chromatographies on DEAE-Sephacel and Sepharose CL-6B. HPLC and electrophoreses on polyacrylamide gel and cellulose acetate membrane revealed the presence of one species of low-sulfated polysaccharide with a relative molecular mass of 20-kDa. Chemical analyses of the polysaccharide showed that it is rich in glucosamine (46%) and neutral sugars (30-34%) with small amounts of sulfates (5.7-6.5%) and glucuronic acid (2.9-3.4%). Ten percent of the glucosamine is sulfated at the amino group. The neutral monosaccharides present are glucose, fucose, and xylose with glucose as the predominant one. It is estimated that the polysaccharide consists of 61-65 molecules of glucosamine (6-7 of which are N-sulfated), 30-35 neutral monosaccharides, 3-4 molecules of glucuronic acid, and 1-3 of fucose and xylose. Isolation and characterization of such a polysaccharide from the extracellular slime layer of S. epidermidis has not been previously reported. Its role to pathogenicity remains to be elucidated.

Bacterial Proteins↗
PubMed1989

Are glycoconjugates and their endogenous receptors involved in the fusion of mononuclear macrophages resulting in multinucleate giant cells? Histochemical and electron microscopic determination of endogenous sugar-binding proteins (lectins) in mononuclear macrophages and multinucleate giant cells appearing in granulomatous foreign body reaction.

Protein-carbohydrate recognition has been found to play an important role in phagocytosis. Labelled (neo)glycoproteins were employed to comparatively analyze the histochemical pattern and ultrastructural localization of endogenous carbohydrate-binding proteins (lectins) of mononuclear macrophages and multinucleate giant cells involved in the granulomatous foreign body reaction. Sugar receptors having an affinity to simple alpha- and beta-galactoside-structures, to alpha-mannose residues, to N-acetylglucosamine, to N-acetylgalactosamine and to glucuronic acid, respectively, were detected in both cell types. However, alpha-fucoside- and beta-xyloside-specific receptors were present only in the mononuclear macrophages. Pronounced differences were seen with labelled, suitably modified glycoproteins, exposing different complex sugar parts with common beta-galactoside-termini. Among the population of multinucleate giant cells, a positive histochemical reaction was observed with mannose-6-phosphate-, galactose-6-phosphate- and glucuronic acid-(BSA-biotin), respectively, only in giant cells in which fusing mononuclear cells were recognizable. This transient expression indicates changes within the profile of endogenous sugar receptors in the stages from fusion to establishment of giant cells. Aside from the diffuse intracytoplasmic distribution of carbohydrate-binding proteins, a prominent accumulation of various types of glycosylated ferritin, used as a marker for electron microscopic evaluation, was ultrastructurally found in membranous subcellular structures and vesicles. This study is a basis for further investigation of the potential involvement of various sugar receptors in the process of macrophage fusion, resulting in multinucleate giant cells of foreign body type, and the process of phagocytosis.

Biopsy↗
PubMed1995

Relationship between the anchor structure of the galactosyl ligand for liposome modification and accumulation in the liver.

Liposomes which have been modified with (8-hexadecanoylamido-3,6-dioxaoctyl)-beta-D-galactose (Gal-t-pa), a straight chain palmitoyl derivative, and are composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), and dicetyl phosphate (DCP) at a ratio of 10:10:1, showed the same accumulation in the liver as the control liposome. Also, liposomes which have been modified with [8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl]-beta-D-gal actoside (Gal-t-psa) showed remarkable accumulation in the liver. The accumulation of liposomes modified with galactose derivatives in the rat liver differed markedly according to the anchor structure. To clarify the cause of this finding, we produced [3H]inulin entrapped [14C]Gal-t-pa modified double label liposomes and evaluated changes in their rat plasma concentration, distribution in the organs, and the in vitro interaction with rat plasma. [14C]Gal-t-pa on the liposome surface bound to serum albumin and was released, resulting in no accumulation in the liver. In addition, sialic acid palmitoyl derivatives and glucuronic acid palmitoyl derivatives behaved similarly. As with the galactose derivatives, they also bound to serum albumin, being released from liposomes. These results suggest that adequate attention should be paid to the anchor structure of the ligand, in order to incorporate a recognition element into liposomes for transport to cells.

Animals↗
PubMed1982

Chemical modifications of Escherichia coli L-asparaginase and their effect on plasma clearance rate and other properties.

Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been modified by succinylation, acetylation and the attachment of N,N-dimethyl-1,3-propanediamine and glucuronic acid. The effect of these modifications on plasma clearance rates in mice and on other properties is compared to the effects of modification with lactose and N-acetylneuraminyl lactose studied previously. The t 1/2 values for the acylated enzyme samples (lower pI) were reduced, succinylated asparaginase sharply and the acetylated enzyme less so. The N,N-dimethyl-1,3-propanediamine-modified samples (increased pI) also had lower t 1/2 values, but samples modified with glucuronic acid (reduced pI) showed little change in clearance time. The main conclusion is that the increased t 1/2 value found in the previous work for N-acetylneuraminyl-lactosylated enzyme is not due to the decreased pI value of the modified enzyme, but must be attributed to interference by N-acetylneuraminyl-lactose residues, directly or indirectly, with the mechanism normally used by the mouse to clear itself of injected E. coli asparaginase.

Acetylation↗
PubMed1986

Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroids in horse urine. I. Detection of testosterone phenylpropionate administrations to equine male castrates.

A gas chromatographic-mass spectrometric (GC-MS) method using three analytes to detect and confirm the administration to equine male castrates of veterinary pro-drugs based upon esters of testosterone is described. The method involves extraction of steroid conjugates from horse urine by C18-bonded cartridges and fractionation into glucuronic acid and sulpho-conjugates by Sephadex LH-20 column chromatography. After deconjugation, the free neutral steroids were partially purified by thin-layer chromatography and following derivatization (methyloxime-trimethylsilyl ether) were analysed by capillary GC-MS in the selected-ion or full-scan mode. Of the three analytes, 5 alpha-androstane-3 beta, 17 alpha-diol could be detected in the glucuronic acid fraction for about ten days and 5 alpha-androstane-3 beta,17 beta-diol and testosterone could be detected in the sulpho-conjugate fraction for up to nineteen days after administration of a single therapeutic dose (50 mg) of testosterone phenylpropionate to cross-bred and thoroughbred castrated male horses. The reasons for development of such a method, its validation and its potential for the detection of neutral metabolites of other veterinary anabolic steroids in horse urine are discussed.

Animals↗
PubMed1989

Exposure of rats to inhalational anesthetics alters the hepatobiliary clearance of cholephilic xenobiotics.

Several recent studies indicate that anesthesia-induced alteration of UDP-glucuronic acid concentrations can affect the rate of xenobiotic glucuronidation by UDP-glucuronosyltransferases. Other data demonstrate that the biliary excretion of several cholephilic drugs is depressed in rats anesthetized with diethyl ether. The present study has examined the effect of 2% halothane, 1.5% isoflurane, 2% enflurane and 3% sevoflurane on the clearance and biliary excretion of acetaminophen, digoxin, phenol red and phenol-3,6-dibromphthalein disulfonate. All volatile anesthetics reduced hepatic UDP-glucuronic acid concentrations 50 to 75%. Biliary excretion of acetaminophen as well as the glucuronide and sulfate conjugates was depressed by all anesthetics for about 1 hr, whereas biliary excretion of the glutathione conjugate was increased during this time. Although total clearance, elimination half-life and steady-state volume of distribution were not altered, biliary clearance of acetaminophen was decreased by 39 to 50%. Formation of the glucuronide conjugate of phenol red and its biliary excretion were depressed by all volatile anesthetics; however, total clearance was increased by 15 to 25% during isoflurane or sevoflurane anesthesia. Total clearance and steady-state volume of distribution of digoxin were decreased only in rats exposed to halothane. There were no changes in biliary excretion. Urinary clearance of digoxin was increased by all volatile anesthetics, whereas biliary clearance was decreased by halothane and enflurane. Biliary excretion, clearance and volume of distribution of phenol-3,6-dibromphthalein disulfonate were not altered by the anesthetics. These data indicate that the hepatobiliary elimination of the glucuronidated metabolites is reduced in rats exposed to volatile anesthetics.

Acetaminophen↗
PubMed2003

Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis.

EXTL2, an alpha1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.

Amino Acid Sequence↗
PubMed1995

A limitation in the use of mass isotopomer distributions to measure gluconeogenesis in fasting humans.

The use of distributions of mass isotopomers in glucose from [U-13C]glycerol to estimate fractional rates of gluconeogenesis was examined. [U-13C]glycerol was infused into normal subjects who ingested acetaminophen and fasted for 60 h. Isotopomer distributions were measured by mass spectrometry in blood glucose and in glucuronic acid from urinary acetaminophen glucuronide. The distributions are incompatible with glucose production solely via gluconeogenesis from a single pool of triose phosphates. Rather, with the assumption of a single enriched triose phosphate pool, the distributions indicate, despite the 60 h of fasting, about as much glucose formation from an unlabeled glucose source as from that pool. Therefore the data indicate cellular heterogeneity in glycerol's metabolism, so that two or more pools with significantly different enrichments were the source of the glucose and glucuronic acid. This heterogeneity is related to much greater concentrations of glycerol in periportal than in pericentral zones of the liver lobule. Beyond evidence for heterogeneity, the findings emphasize a limitation in applying analyses of mass isotopomer distributions to measure polymer biosynthesis in the presence of heterogeneity in the precursor pool.

Acetaminophen↗
PubMed1977

Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases.

The bilrubin-IXalpha conjugates in bile and the activities of bilirubin-IX alpha--UDP-glycosyltransferases in liver and kidney were determined for ten species of mammals and for the chicken. 1. In the mammalian species, bilirubin-IX alpha glucuronide was the predominant bile pigment. Excretion of neutral glycosides was unimportant, except in the cat, the mouse, the rabbit and the dog, where glucose and xylose represented 12--41% of total conjugating groups bound to bilirubin-IX alpha. In chicken bile, glucoside and glucuronide conjugates were of equal importance. They probably represent only a small fraction of the total bile pigment. 2. The transferase activities in liver showed pronounced species variation. This was also apparent with regard to activation by digitonin, pH optimum and relative activities of transferases acting on either UDP-glucuronic acid or neutral UDP-sugars. 3. Man, the dog, the cat and the rat excrete bilirubin-IX alpha largely as diconjugated derivatives. In general, diconjugated bilirubin-IX alpha could also be synthesized in vitro with liver homogenate, bilirubin-IX alpha and UDP-sugar. In contrast, for the other species examined, bilirubin pigments consisted predominantly of monoconjugated bilirubin-IX alpha. Synthesis in vitro with UDP-glucuronic acid, UDP-glucose or UDP-xylose as the sugar donor led exclusively to the formation of monoconjugated bilirubin-IX alpha. 4. The transferase activities in the kidney were restricted to the cortex and were important only for the rat and the dog. No activity at all could be detected for several species, including man. 5. Comparison of the transferase activities in liver with reported values of the maximal rate of excretion in bile suggests a close linkage between conjugation and biliary secretion of bilirubin-IX alpha.

Animals↗
PubMed2002

Formation of a structurally novel, serial diglucuronide of 4-hydroxybiphenyl by further glucuronidation of a monoglucuronide in dog liver microsomes.

Incubation of 4-hydroxybiphenyl (p-phenylphenol) in the presence of UDP-glucuronic acid (UDPGA) with liver microsomes from male and female dogs produced a more polar metabolite peak than a simultaneously produced peak of 4-hydroxybiphenyl monoglucuronide in the high performance liquid chromatography (HPLC) chromatogram. Tandem mass spectrometry (MS/MS) and two-dimensional nuclear magnetic resonance (NMR) analyses revealed this polar metabolite as a 4-hydroxybiphenyl diglucuronide having a beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl moiety, where the two glucuronic acids are connected directly at the 1''-->2' position. Liver microsomes from Sprague-Dawley rat, cynomolgus monkey and human, converted 4-hydroxybiphenyl only to the monoglucuronide, suggesting that there is a dog UDP-glucuronosyltransferase (UGT), with a wider substrate specificity capable of glucuronidating 4-hydroxybiphenyl monoglucuronide to the diglucuronide.

Journal Article↗
PubMed2000

[Study on hydroxylated metabolites of benproperine in human urine].

AIM: To investigate the hydroxylation process of benproperine in humans. METHODS: After an oral administration of 60 mg benproperine to ten healthy male volunteers, urine samples collected within 0-24 h were extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry. A microbial transformation of benproperine combined with semi-preparative HPLC was used to get two reference substances of the hydroxylated metabolites, and their structures were then elucidated by NMR. Furthermore, the structures of conjugated metabolites were speculated based on their characteristics in MS fragmentation. RESULTS: Five hydroxylated metabolites of benproperine and some of their conjugates with endogenous glucuronic acid or sulfuric acid were found in urine of volunteers after the dose. The structures of two metabolites were identified as 4"-hydroxybenproperine and 4"'-hydroxybenproperine by comparison of HPLC retention times and mass spectra with those of authentics obtained from the microbial transformation. CONCLUSION: Hydroxylation of benproperine occurs preferentially at para-position of the alkoxyl group in the aromatic ring. The hydroxylated metabolites of benproperine in human urine mainly exist as their glucuronic acid or sulfuric acid conjugates.

Adult↗
PubMed2003

Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications.

Alpha-glucuronidases cleave the alpha-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the alpha-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the alpha-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp(364) and Glu(392), activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu(285) cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu(285) is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.

Geobacter↗
PubMed1985

The disaccharide effect of sucrose feeding on glucuronide excretion and bile concentration of injected phenolphthalein in guinea pigs.

The hypothesis tested was that feeding guinea pigs sucrose produces a more rapid concentration in the bile and excretion in the feces and urine of substances catabolized by the liver than does feeding invert sugar (50:50 mixture of glucose and fructose). Fifty male guinea pigs of the Hartley strain were divided into two groups of 25 animals each and fed for 4 wk repelleted nonpurified diet with 20% of total energy provided by sucrose or invert sugar. At the end of 4 wk all 50 animals were injected i.p. with a dose of 15 mg/kg body weight of phenolphthalein. Phenolphthalein is excreted almost quantitatively in feces. After injection all guinea pigs were housed in metabolism cages. Urine and feces were recovered and analyzed for free glucuronic acid and glucuronide content by a modified naphthoresorcinol procedure over 24 h. Guinea pigs fed sucrose produced more urine than those fed invert sugar, although there was no difference in water intake. After 24 h 15 animals in each group were killed, and the bile was sampled from their gall bladders to determine its phenolphthalein content. The remaining 10 animals in each group were held three additional days when they were killed and their bile was sampled to determine its phenolphthalein content. All biliary phenolphthalein was in conjugated form. Guinea pigs fed sucrose had less free glucuronic acid in their feces than those fed invert sugar. Feeding sucrose resulted in a higher bile conjugated phenolphthalein content 4 d after injection than did feeding invert sugar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The inducibility and ontogeny of rat liver UDP-glucuronyltransferase toward furosemide.

Furosemide (F) conjugation with glucuronic acid is the main pathway of F metabolism in humans and experimental animals. In order to study rat liver microsomal UDP-glucuronyltransferase (UDP-GT) activity towards F we developed an in vitro assay in which the conjugation product, furosemide 1-0-acyl glucuronide (FG) was separated and quantitatively determined by reverse phase high pressure liquid chromatography. The optimal conditions of the reaction were established and the apparent Km for F and UDP-glucuronic acid (UDPGA) were 0.22 and 1.76 mM, respectively. Substrate inhibition of UDP-GT toward F occurred at F concentrations higher than 1.5 mM. Developmental changes in F glucuronidation were compared to the ontogeny of UDP GT activity toward two other acceptors, 1-naphthol and estrone that are known to have different patterns of maturation. F glucuronidation was 26% of adult activity at 18 days of gestation, reached 48% at birth and gradually increased to 250% of adult activity at 22 days of age. Glucuronidation of 1-naphthol and estrone attained 87% and 44% of adult activity at 22 days of gestation, 37% and 66% in six-day-old rats and 100% and 427% of adult activity in 22-day-old rats, respectively. The effect of 3-methylcholanthrene (3-MC), phenobarbital (PB) and pregnenolone-16 alpha-carbonitrile (PCN) on F UDP-GT was studied and compared to their effect on 1-naphthol and estrone glucuronidation. PB, 3-MC and PCN increased F-UDP-GT activity to 208%, 282% and 342% of vehicle-treated animals, respectively, while F pretreatment did not affect the conjugation of F. In comparison, 1-naphthol glucuronidation was preferentially induced by 3-MC (4.4-fold of control) while estrone glucuronidation was induced by PB and PCN (4.9- and 2.5-fold of control, respectively). These studies suggest that several forms of UDP-GT activities, which differ in their ontogeny and inducibility patterns, are involved in the glucuronidation of F in vitro.

Animals↗
PubMed2003

Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose.

In Escherichia coli and Salmonella typhimurium, addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate group(s) of lipid A is required for resistance to polymyxin and cationic antimicrobial peptides. We have proposed previously (Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. H. (2002) J. Biol. Chem. 277, 2886-2896) a pathway for l-Ara4N biosynthesis that begins with the ArnA-catalyzed C-4" oxidation and C-6" decarboxylation of UDP-glucuronic acid, followed by the C-4" transamination of the product to generate the novel sugar nucleotide UDP-l-Ara4N. We now show that ArnB (PmrH) encodes the relevant aminotransferase. ArnB was overexpressed using a T7lac promoter-driven construct and shown to catalyze the reversible transfer of the amino group from glutamate to the acceptor, uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate), the intermediate that is synthesized by ArnA from UDP-glucuronic acid. A 1.7-mg sample of the putative UDP-l-Ara4N product generated in vitro was purified by ion exchange chromatography, and its structure was confirmed by 1H and 13C NMR spectroscopy. ArnB, which is a cytoplasmic protein, was purified to homogeneity from an overproducing strain of E. coli and shown to contain a pyridoxal phosphate cofactor, as judged by ultraviolet/visible spectrophotometry. The pyridoxal phosphate was converted to the pyridoxamine form in the presence of excess glutamate. A simple quantitative radiochemical assay was developed for ArnB, which can be used to assay the enzyme either in the forward or the reverse direction. The enzyme is highly selective for glutamate as the amine donor, but the equilibrium constant in the direction of UDP-l-Ara4N formation is unfavorable (approximately 0.1). ArnB is a member of a very large family of aminotransferases, but closely related ArnB orthologs are present only in those bacteria capable of synthesizing lipid A species modified with the l-Ara4N moiety.

Amino Sugars↗
PubMed1994

Metabolism of coumarin and 7-ethoxycoumarin by rat, mouse, guinea pig, cynomolgus monkey and human precision-cut liver slices.

1. The metabolism of 50 microM 7-ethoxycoumarin and 50 microM [3-14C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man. 2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species. 3. [3-14C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites. 4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-14C]coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species. 5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-14C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earle's balanced salt solution). 6. These results demonstrate that precision-cut liver slices are a valuable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.

Animals↗