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Three-dimensional X-ray diffraction data were used to determine the crystal structure of alpha-D-glucuronate CaBr times 3H20, a model system for investigating the factors involved in the binding of calcium ions to D-glucuronate residues of oligo-and poly-saccharides. Crystals of the salt are monoclinic, space group P21, having a = 6.410 (1), b = 10.784 (2), c = 8.879 (1) A, betta = 92.07 (1)degrees, and Z = 2. Instensity data for 1082 reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least squares to R = 0.025. The absolute configuration was confirmed by anomalous-dispersion effects. An outstanding feature of the crystal packing is the interaction of D-glucuronate anions with calcium ions. The calcium ion is coordinated to three symmetry-related D-glucuronate anions and to two water molecules. The D-glucuronate anion binds calcium cations through three chelation sites: one that involves a carboxyl-oxygen atom combined with O-5; one that includes the second carboxyl-oxygen atom acting in concert with O-4, and one composed of the O-1-O-2 pair of hydroxyl groups.
A new senitive and specific micromethod for determination of UDP-glucuronic acid is described Extracts from 2.5 mg of liver are incubated with twice washed guinea pig microsomes (as a source of UDP-glucuronyl transferase) and [14C]p-nitrophenol. The content of UDP-glucuronic acid is calculated from the [14C]p-nitrophenyl glucuronide/[14C]p-nitrophenol radioactivity ratio and the known amount of introduced [14C]p-nitrophenol. These values are corrected for losses of UDP-glucuronic acid by a calibration experiment, containing in addition to the aforementioned constituents, a known amount of UDP-glucuronic acid. The mean concentration of UDP-glucuronic acid in mice liver was found to be 171 mumol/kg wet weight.
The biosynthesis of glucuronosyl diacylglycerol from UDP-glucuronate and diacylglycerol is catalyzed by an enzyme found in both the 34,800 X g supernatant and particulate preparations from disrupted Pseudomonas diminuta (ATCC 11586). UDP-glucuronate served as the glucuronosyl donor and could not be replaced by glucuronic acid, glucuronate-1-phosphate, and a number of nucleotide-linked sugars. The maximum velocity was estimated to be 19 nmol of glucuronosyl diacylglycerol synthesized/h/mg of protein in the presence of the 34,800 X g particulate enzyme and 63 nmol/h/mg of protein with the 34,800 X g supernatant preparation. The apparent Km for UDP-glucuronate was 4.2 micronM for supernatant and 4.4 to 6.0 micronM for particulate preparations. The biosynthesis of glucuronosyl diacylglycerol in vitro, was strongly dependent upon exogenous diacylglycerols containing unsaturated and shorter chain fatty acids. The enzymatic activity was very heat-labile and lost about 80% of the initial rate of synthesis after preincubation for 5 min at 37 degrees. The reaction was stimulated by 14.7 mM Triton X-100 and had an optimal pH of 7.1 and an ionic strength of 0.2 M. Divalent cations were not required.
The extracellular, bacterial polysaccharides from seven Rhizobium strains have been submitted to partial hydrolysis with acid. Several neutral oligosaccharides, some containing pyruvic acid, were isolated together with D-glucuronic acid-containing oligosaccharides. The polysaccharide from Rh. meliloti did not contain glucuronic acid. For the other six strains, the following components were characterized: 4-O-(beta-D-glucopyranosyluronic acid)-D-glucuronic acid, 4-O-(beta-D-glucopyranosyluronic acid)-D-glucose, and O-(beta-D-glucopyranosyluronic acid)-(1leads to4)-O-(beta-D-glucopyranosyluronic acid)-(1leads to4)-D-glucose. These results indicate the presence of chains containing two beta-(1leads to4)-linked D-glucuronic acid residues, beta-linked to D-glucose at position 4.
The development and relative contribution of hepatic bilirubin conjugation with glucuronic acid, xylose, and glucose was studied in vitro in newborn rats 1-20 days old. In adult control rats, 75% of the conjugates formed were with glucuronic acid, whereas in 1-day-old newborns, only 50% of the conjugates were with glucuronic acid (P less than 0.02) while xylose and glucose conjugates of bilirubin together were equal to that of glucuronic acid. By day 4, total conjugating capacity increased to adult levels and a mature pattern of distribution. In response to phenobarbital treatment, xylose and glucose conjugation increased 4 days earlier than glucuronide conjugation and maximal induction occurred 8 days sooner for nonglucuronide conjugation than for glucuronide.
A new method is reported for the quantitative determination of glucuronic and galacturonic acid, which is based on spectrophotometric measurement of NADH. The NAD-linked oxidation of the uronic acids to the corresponding dicarboxylic acids is measured in the presence of uronic acid dehydrogenase. This enzyme was isolated from Pseudomonas syringae. The test is highly specific for glucuronic and galacturonic acid and permits the exact determination of free and conjugated glucuronic acid. This enzymatic determination of glucuronic is the most sensitive method available today.
The mucopolysaccharide, hyaluronic acid, is an important component of both mammals and pathogenic streptococci. This high molecular weight polymer is synthesized by a membrane-associated, multisubunit hyaluronate synthase which utilizes UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Using the photoaffinity probe, [beta-32P]5-azido-UDP-glucuronic acid, three streptococcal membrane proteins (42, 33, and 27 kDa) specifically photoincorporated this probe. Labeling of these proteins was enhanced in the presence of UDP-N-acetylglucosamine, whereas UDP-galactose or UDP-glucose had no effect on incorporation. UDP-glucuronic acid inhibited the labeling of the three proteins in a dose-dependent manner. Detergent-solubilized membrane proteins from transposon-inactivated hyaluronic acid capsule mutants no longer incorporated the probe. This was also the case when membranes from stationary phase organisms were tested. Finally, glucuronic acid no longer was incorporated into high molecular weight hyaluronic acid with either the mutant or stationary phase preparations. Further biochemical analysis will be required to demonstrate the exact role each of the proteins play in hyaluronic acid biosynthesis.
UDP-glucuronate carboxy-lyase has been demonstrated in chick chondrocytes in tissue culture. It occurs in the particulate fraction, and its activity is stimulated by exogenous NAD. The enzyme is allosterically activated by UDP-glucuronate and inhibited by UDP-xylose, n Values of 2.8 indicate positive cooperativity of at least three interacting sites on the enzyme. These data suggest that UDP-xylose concentration in chondrocytes is regulated by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. Activity levels of the enzyme during growth of the cells peak towards mid-log phase and decline thereafter, closely paralleling levels of chondroitin sulfate glycosyltransferases determined previously (Schwartz, N. B. (1976) J. Biol. Chem. 251, 3346-3351). Thus, it appears that during chondrocyte development a common mechanism governs induction of glycosyltransferases and of UDP-glucuronate carboxy-lyase.
The black yeast-like fungus NRRL YB-4163, now tentatively identified as Rhinocladiella elatior Mangenot, has been found to produce an extracellular microbial polysaccharide composed mainly of 2-acetamido-2-deoxy-D-glucuronic acid residues. Polysaccharide (PS) YB-4163, when isolated in good yield as the neutral potassium salt, dissolves readily in water to produce extremely viscous solutions, which form stable foams and emulsions. By depolymerizing PS YB-4163 with [14C]methanol-HCl, the polysaccharide can be both identified and quantitated radiochemically by determining the individual [14C]methyl glycosides after their separation by paper chromatography. When the methyl glycosides of PS YB-4163 were reduced with NaB3H4, only the methyl glycosides of 2-acetamido-2-deoxy-D-[6-3H]glucose were found. Analysis of the monosaccharide released from carboxyl-reduced PS YB-4163 by acid hydrolysis or methanolysis also showed 2-acetamido-2-deoxy-D-glucuronic acid to be the main constituent. Previously, the only polysaccharides known to be composed entirely or hexosaminuronic acid have been cellular products from pathogens. Of these, the antigenic polysaccharide (SPSA) from Staphylococcus aureus is composed entirely of 2-amino-2-deoxy-D-glucuronic acid, but its amino groups are substituted equally with acetyl and N-acetylalanyl groups. The specific optical rotation of PS YB-4163, [alpha]20D -75 degrees (c 0.5, water), is similar to that of SPSA (-91 degrees), and suggests beta-D-linkages that must be either (1 leads to 3) or (1 leads to 4).
The structure of dermatan [35S]sulphate-chondroitin [35S]sulphate copolymers synthesized and secreted by fibroblasts in culture was studied. 35S-labelled glycosaminoglycans were isolated from the medium, a trypsin digest of the cells and the cell residue after 72h of 35SO42-incorporation. The galactosaminoglycan component (dermatan sulphatechondroitin sulphate copolymers) was isolated and subjected to various degradation procedures including digestion with testicular hyaluronidase, chondroitinase-AC and-ABC and periodate oxidation followed by alkaline elimination. The galactosaminoglycans from the various sources displayed significant structural differences with regard to the distribution of various repeating units, i.e. IdUA-GalNAc-SO4 (L-iduronic acid-N-acetyl-galactosamine sulphate), GlcUA-GalNAc-SO4 (D-glucuronic acid-N-acetylgalactosamine-sulphate) and IdUA(-SO4)-GalNAc (L-iduronosulphate-N-acetylgalactosamine). The galactosaminoglycans of the cell residue contained larger amounts of IdUA-GalNAc-SO4 than did those isolated from the medium or those released by trypsin. In contrast, the glycans from the latter 2 sources contained large proportions of periodate-resistant repeat periods [GlcUA-GalNAc-SO4 and IdUA(-SO4)-GalNAc]. Periods containing L-iduronic acid sulphate were particularly prominent in copolymers found in the medium. Kinetic studies indicated that the 35S-labelled glycosaminoglycan of the cell residue accumulated radioactivity more slowly than did the glycans of other fractions, indicating that the material remaining with the cells was not exclusively a precursor of the secreted polymers. The presence of copolymers rich in glucuronic acid or iduronic acid sulphate residues in the soluble fractions may be the result of selective secretion from the cells. Alternatively, extracellular, polymer-level modifications such as C-5 inversion of L-iduronic acid to D-glucuronic acid, or sulphate rearrangements, would yield similar results.
A new acidic sphingoglycolipid has been isolated from a Gram-negative, glucose-non-fermentative (obligatory aerobic) bacterium, Flavobacterium devorans ATCC 10829, by thin-layer chromatography on silica gel after mild alkaline hydrolysis of the cellular lipids. Chemical degradation studies, thin-layer chromatographic behavior, IR and mass-spectrometric analysis of the original and reduced glycolipid with LiA1H4 revealed that the lipid contained glucuronic acid, long-chain bases, and fatty acids in a molar ratio of approximately 1:1:1. The major long-chain bases were identified by gas chromatography-mass spectrometry as dihydrosphingosine (d-18 :0) and longer homologues, while the N-acyl group was exclusively 2-hydroxy myristic acid. The most probable structure of this glycolipid appeared to be a ceramide glucuronic acid (N-acyl dihydrosphingosine 1-glucuronic acid).
The urinary levels of D-glucaric acid, which is an index of hepatic microsome induction, and the excretion of glucuronic acid and porphyrins were measured in nine patients with Porphyria Cutanea Tarda (PCT) and twelve normal controls. The excretion of D-glucaric acid and glucuronic acid were respectively 3.5 and two times higher in PCT patients compared to controls. A statistical correlation could be demonstrated between urinary excretion of total porphyrins with that of glucaric and glucuronic acids. These findings indicate that microsomal function and porphyrin metabolic derangement are strictly related in PCT.
DEAE-cellulose chromatography of partially purified preparations of UDP-glucuronate carboxy-lyase from wheat germ results in the separation of two forms of the enzyme. Both are fully active in the absence of added DPN, have indistinguishable molecular weights (210,000), but differ in charge and kinetic properties. Both are cooperatively activated by UDP-glucuronate, however Enzyme 1 is activated at lower concentrations than Enzyme 2. At low substrate concentrations (less than or equal to 5 micron), both enzymes are activated by UDP-glucose, 2 mM concentrations of activator increasing the activity of Enzyme 1 2-fold and of Enzyme 2 2.5-fold. UDP-xylose allosterically inhibits both enzymes. At substrate concentrations equal to the apparent Km values, inhibition of Enzyme 1 is much greater than that of Enzyme 2 (83 and 28% at 0.33 mM inhibitor concentration). The data suggest that synthesis of UDP-xylose is controlled both by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. The existence of a "more active" and a "less active" species of the enzyme suggests the possibility of two interconvertible forms of the same protein and the involvement of such interconversion in further regulation of UDP-xylose biosynthesis. However it is equally possible that both represent true isoenzymes.
Hepatic enzymes connected with the formation and metabolism of free D-glucuronic acid were affected in rats after treatment with disulfiram or diethyldithiocarbamate (300 mg/kg, intragastrically, per day, 4 X). The activities of UDPglucose dehydrogenase, UDPglucuronic acid pyrophosphatase, UDPglucuronosyltransferase and L-gulonate dehydrogenase were enhanced, while those of glucose-6-phosphate dehydrogenase, beta-glucuronidase and D-glucuronolactone dehydrogenase were inhibited. These changes were more pronounced with disulfiram than diethyldithiocarbamate. Treatment with phenobarbital (80 mg/kg, i.p., per day, 4 X) enhanced UDP glucuronosyl-transferase, but brought about different effects on the other enzymes. Concurrent administration of phenobarbital with disulfiram or diethyldithiocarbamate led to potentiation or antagonism of the primary effects of each compound when given alone. The results suggest that activation of the D-glucuronic acid pathway may proceed in various ways, and that it is not necessarily followed by a simultaneous induction of the microsomal mixed-function oxygenase activity.
Localization of glucuronic acid-containing glycosaminoglycans in the gerbil utricle was examined, using a hyaluronidase-gold labeling technique with special emphasis on the otoconia. Otoconia and the gelatinous layer of the otoconial membrane were strongly labeled by hyaluronidase-gold. The secretory granules in supporting cells were also strongly labeled, suggesting that the organic matrix of otoconia is secreted from the supporting cells. Otoconia seem to lose labeling while they degenerated. The degenerating otoconia were observed to be absorbed into dark cells. Glucuronic acid-containing glycosaminoglycans occur in otoconia. These glycosaminoglycans may play a crucial role in the formation and degeneration of otoconia.
Chondroitin ABC and AC lyases split hexosaminidic linkages in galactosaminoglycans and hyaluronic acid. Even-numbered oligosaccharides from hyaluronic acid with either D-glucuronic acid or N-acetylglucosamine in non-reducing position were used, prior to and after reduction with sodium borohydride, as substrates for chondroitin ABC and AC lyases. These substrates allowed elucidation of the effects of the nearest neighborhood of the bond to be split on the action of the enzymes. The results indicate that chondroitin ABC lyase acts strictly as an endolyase towards hyaluronate and requires the presence of a disaccharide in both reducing and non-reducing positions of the endohexosaminidic bond to be split. None of the hexosaminidic bonds of the tetrasaccharide GlcNAc-GlcUA-GlcNAc-GlcUA is split by chondroitin ABC lyase. In contrast chondroitin AC lyase acts also as an exoglycosidase towards hyaluronate and recognizes only the amino sugar and the uronic acid residue that are linked via the hexosaminidic bond which is split. Thus, the N-acetylglucosamine and glucuronic acid residues at both ends of a tetrasaccharide with the structure GlcNAc-GlcUA-GlcNAc-GlcUA are liberated.
1. Urinary levels of xylitol and glucuronic, glucaric, gulonic and ascorbic acids were measured in the rat, rabbit, guinea-pig and marmoset by an improved g.l.c. technique. 2. Administration of a compound (2-methylbenzanilide) known to be conjugated and excreted as a beta-glucuronide had some effect on the output of these compounds of the glucuronic acid pathway in all four species, and caused a significant decrease in gulonic acid in the rat.