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PubMed1982

The discovery of a lipid-linked glucuronide and its synthesis by chicken liver.

Upon incubation with uridine diphosphate-[14C]glucuronic acid, membrane fractions from adult and phenobarbital-induced embryonic liver synthesize a single glucuronide, which is soluble in chloroform:methanol (2:1). The compound is completely hydrolyzed and glucuronic acid released by either mild acid or beta-glucuronidase, whereas mild base hydrolysis results in a mixture of glucuronic acid and glucuronic acid-1,2-cyclic phosphate. These data and the behavior of the lipid-linked glucuronide on DEAE-cellulose chromatography indicate that the compound contains a monophosphate diester of glucuronic acid, which is beta-linked to a lipid. The synthesis of the lipid-linked glucuronide in uninduced normal embryonic liver is very low (5-15 pmol product/mg/5 min) at all developmental ages up to hatching, but the introduction of phenobarbital into the air space of a 9-10-day-old embryo causes a premature increase of activity (75-150 pmol products/mg/5 min) within 7 days. The glucuronyltransferase in adult and induced embryonic liver has a Km for UDPGlcUA of 0.17 x 10(-3) M and a broad pH optimum between pH 6 and 7. Glucuronic acid is released from the lipid-linked glucuronide by a beta-glucuronidase in liver that is active at neutral pH and is not inhibited by saccharolactone. This glycosidase activity appears, therefore, to be distinct from the previously characterized lysosomal beta-glucuronidase. Fractionation of adult chicken liver membranes by differential centrifugation indicates that over 70% of the glucuronyltransferase is associated with the nuclear and mitochondrial fractions. The endogenous beta-glucuronidase capable of hydrolyzing the lipid-linked glucuronide was not separated from the glucuronyl-transferase activity during fractionation. The data available suggests that the lipid-linked glucuronide is involved directly in the generation of free glucuronic acid for further metabolism.

Animals↗
PubMed1994

Serum factors, growth factors and UDP-sugar metabolism in bovine articular cartilage chondrocytes.

1. The effect of different batches of fetal bovine serum and of growth factors on [35S]sulphate incorporation into glycosaminoglycans and on UDP-sugar pools in explant cultures of bovine articular cartilage was investigated. 2. [35S]Sulphate incorporation was variably stimulated between 1.2- and 3.5-fold by four different batches of serum. The UDP-glucuronate pool size expanded 4.3-6.5-fold in the presence of serum, even in those cultures in which little stimulation of [35S]sulphate incorporation occurred. The UDP-N-acetylhexosamine and UDP-hexose pools expanded by about 1.5- and 2.0-fold respectively in the presence of serum. UDP-xylose was not detected. 3. Equilibrium-labelling and pulse-chase experiments with D-[1-3H]glucose indicated that the rate of flux through the UDP-sugar pools was unaffected by serum. UDP-hexose, UDP-N-acetylhexosamine and UDP-glucuronate have approximate half-lives (t1/2) of 7, 12 and 3-4 min respectively. At equilibrium, the 3H specific activities of UDP-hexose and UDP-N-acetylhexosamine were very similar but that for the UDP-glucuronate pool was much higher, especially in serum-supplemented cultures. The results suggest that UDP-glucuronate synthesis occurs via a pathway which is independent of the main UDP-hexose pathway. 4. Supplementing cultures with heat-treated serum had no effect on the serum-induced expansion of UDP-sugar pools but stimulation of [35S]sulphate incorporation into glycosaminoglycans was 50% lower than for native serum. Acid-treated serum promoted a 2-fold expansion of the UDP-glucuronate and UDP-N-acetylhexosamine pool over that obtained with native serum but was 20% less effective in stimulating [35S]sulphate incorporation than the latter. Prior dialysis of serum had no effect on its modulatory action on either [35S]sulphate incorporation or on the size of UDP-sugar pools. 5. Insulin-like growth factor 1 (IGF-1), transforming growth factor beta-1 (TGF beta-1), platelet-derived growth factor (PDGF) (BB homodimer) and epidermal growth factor (EGF) all stimulated [35S]sulphate incorporation into glycosaminoglycans as expected. The UDP-glucuronate pool expanded by 1.5- and 2.0-fold in the presence of IGF-1 and TGF beta-1 respectively, and by about 1.8-fold in the presence of PDGF or EGF. None of the factors investigated, or combinations of IGF-1 and TGF beta-1 or IGF-1 and EGF, stimulated expansion of the UDP-glucuronate pool to the same extent as native serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Directly energized uptake of beta-estradiol 17-(beta-D-glucuronide) in plant vacuoles is strongly stimulated by glutathione conjugates.

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species. Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively. Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake. E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM). The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide. Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM). Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G. Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective. The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump. GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected. By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump. These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.

Adenosine Triphosphate↗
PubMed1987

Hexuronate catabolism in Erwinia chrysanthemi.

In the phytopathogenic enterobacterium Erwinia chrysanthemi, the catabolism of hexuronates is linked to the degradation of pectic polymers. We isolated Mu lac insertions in each gene of the hexuronate pathway and used genetic fusions with lacZ (the beta-galactosidase gene of Escherichia coli) to study the regulation of this pathway. Three independent regulatory genes (exuR, uxuR, and kdgR) were found. Galacturonate and glucuronate were converted into 2-keto-3-deoxygluconate (KDG) by separate three-step pathways encoded by the uxaC, uxaB, and uxaA genes and the uxaC, uxuB, and uxuA genes, respectively. The two aldohexuronates entered the cell by a specific transport system, encoded by exuT. Wild-type strain 3937 was unable to use glucuronate as a carbon source since glucuronate was unable to induce the exuT expression. Mutants able to use glucuronate possessed an inactivated exuR gene. The product of the regulatory gene exuR negatively controlled the expression of exuT, uxaC, uxaB, and uxaA, which was inducible in the presence of galacturonate. The two genes specifically involved in glucuronate catabolism, uxuA and uxuB, formed two independent transcriptional units regulated separately, uxuB expression was not inducible, whereas uxuA expression was induced in the presence of glucuronate and controlled by the uxuR product. KDG, the common end product of both pathways, is cleaved by the kdgK and kdgA gene products. KDG enters the cell by a specific transport system, encoded by kdgT. The regulatory gene kdgR controlled the expression of kdgT, kdgK, and kdgA and partially that of the pel genes encoding pectate-lyases. The real inducer of pectate-lyase synthesis, originating from catabolism of galacturonate or glucuronate, appeared to be KDG. The genes of E. chrysanthemi affecting hexuronate catabolism are separated into six independent transcriptional units exuT, uxaCBA, uxuA, uxuB, kdgK, and kdgA, but only three gene clusters were localized on the genetic map: exuT-uxaCBA, uxuA-uxuB-kdgK, and kdgA-exuR.

Crosses, Genetic↗
PubMed1992

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.

Animals↗
PubMed1980

Structural studies on heparan sulphates. Characterization of oligosaccharides; obtained by periodate oxidation and alkaline elimination.

Three heparan sulphate fractions were subjected to degradation by periodate oxidation and alkaline elimination. The starting materials were one low-sulphated fraction rich in glucuronic acid and N-acetylglucosamine (I), one fraction (IV) that was medium-sulphated, contained similar proportions of N-acetyl and N-sulphate as well as of glucuronic and iduronic acid and one fraction (V) that was oversulphated, contained an excess of N-sulphate and was rich in iduronic acid. Selective periodate oxidation of glucuronic acid residues within N-acetylated regions followed by scission in alkali yielded three categories of fragments that were isolated by gel and ion-exchange chromatography. (a) N-Acetylglucosamine-R compounds where R is the remnant of an oxidised and degraded glucuronic acid residue. (b) Medium-sized oligosaccharides, of the general structure glucosamine-(glycuronic acid-glucosamine)n-R where n = 1--4, contained largely glucuronic acid associated with glucosamines that were N-sulphated, N-acetylated or unsubstituted. In saccharides where n = 4 indications of an alternating arrangement of N-sulphate and N-acetyl groups as well as of iduronic and glucuronic acid residues were obtained. The glucuronic acid residues of the oligosaccharide fragments were susceptible to reoxidation with periodate. The smaller saccharides were not depolymerised by HNO2 despite the presence of N-sulphate groups. (c) Longer oligosaccharide fragments (n greater than or equal to 5) that were highly sulphated and contained heparin-like repeating units, i.e. iduronic acid--glucosamine-N-sulphate with ester-sulphate on both sugars. These oligosaccharides were susceptible to deaminative cleavage.

Animals↗
PubMed1989

The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.

Acetyl Coenzyme A↗
PubMed1980

Kinetic studies on NADPH-linked aldehyde reductase from human liver.

The mechanism of D-glucuronate reduction by human liver NADPH-dependent aldehyde reductase was investigated. At pH 7.4 the Km values for NADPH, NADP+, D-glucuronate and L-gulonate were 2.2 microM, 6 microM, 3.2 mM and 6 mM, respectively. Product inhibition studies in the forward direction (reduction of glucuronate) gave a competitive pattern for the inhibition of NADPH oxidation by NADP+ and non-competitive patterns for the other three inhibitions. In the backward direction all patterns appeared to be competitive. Deuterium isotope effects were dependent on the concentration of D-glucuronate and decreased to unity at infinite concentrations of D-glucuronate. Our findings suggest for aldehyde reductase a kinetic mechanism with sequential ordered binding of NADPH and D-glucuronate and random dissociation of NADP+ and L-gulonate.

Aldehyde Oxidoreductases↗
PubMed1992

Nonenzymic glycation of albumin by acyl glucuronides in vitro. Comparison of reactions with reducing sugars.

Acyl glucuronides are ubiquitous metabolites formed from acidic xenobiotics and endogenous compounds, such as bilirubin. Previous studies indicated that the covalent binding of acyl glucuronides to proteins occurs via an imine intermediate in a manner analogous to the glycation of proteins via reducing sugars. When glucuronic acid was incubated in solution with albumin, it formed 10 and 4 times more fluorescent, Maillard reaction products with albumin after 25 days than did glucose or fructose, respectively. However, radiolabeled glucuronic acid exhibited less covalent binding to albumin than either glucose or fructose. Circular dichroism measurements indicate that glucuronic acid is about 0.02% open chain form with exposure of the reactive aldehyde, whereas fructose and glucose have 2 and 0.0026% present in solution as the open chain; thus, differences in reactivity of the reducing sugars were not correlated with exposure of the free aldehyde. Methyl glucuronate formed little fluorescent product with albumin, suggesting that the C-6 carboxylate of glucuronic acid may facilitate the reactions after covalent binding that lead to the formation of fluorescent products. When acyl glucuronide metabolites of two previously marketed acidic drugs, zomepirac and suprofen, were incubated with albumin at a concentration of 2.5 mM, more fluorescent product was formed than by 500 mM glucose. Reversible binding of the acyl glucuronides to albumin was 60-90%, but almost zero for the free reducing sugars, which indicates that reversible binding may explain the enhanced reactivity of the acyl glucuronides in forming fluorescent products with albumin. These results indicate that acyl glucuronides are reactive metabolites that may cause significant glycation of proteins with glucuronic acid in vivo.

Animals↗
PubMed2001

Dermatan sulfates of normal and scarred fascia.

We evaluated the composition of dermatan sulfates (DS) derived from 23 samples of normal and 23 samples of scarred fascia lata. We analyzed the molecular weight of intact DS chains and the length of chain regions comprising: (1) clusters of L-iduronate-containing disaccharides ("iduronic sections"); (2) clusters of D-glucuronate-containing disaccharides ("glucuronic sections"); and (3) copolymeric sections with both types of disaccharides. A portion of scarred fascia DS chains demonstrated higher molecular weight compared with those from normal tissue. Most disaccharides of DS chains derived from both fascia types form copolymeric segments - heterogeneous in size - with alternatively distributed single disaccharides with glucuronic residues and mainly single ones with iduronate. Only a small number of disaccharides form "glucuronic sections" of heterogeneous size or short "iduronic sections". However, the scarred fascia DS chains demonstrate an increased content of shorter "glucuronic sections" and shorter, often oversulfated, copolymeric segments. It seems that in normal fascia, the DS chain type with a single, long copolymeric region and a single, shorter "glucuronic section" is predominant, while in scarred tissue an increase in multidomain DS chain content may occur.

Adult↗
PubMed1976

Defective function of a microsomal UDP-glucuronyltransferase in Gunn rats.

The kinetic parameters of the p-nitrophenol-metabolizing form of UDP-glucuronyltransferase [-UDPglucuronosyltransferase; UDPglucuronate beta-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] have been compared in liver microsomes from the Gunn strain of rat and from normal; Wistar rats. The abnormally low rate of glucuronidation of p-nitrophenol in the Gunn rats, as compared with Wistar rats, is due to decreased affinity of UDP-glucuronyltransferase for UDP-glucuronic acid. Activities at Vmax and the Michaelis constant for p-nitrophenol, KPNP, of UDP-glucuronyltransferase are the same for enzyme from either strain of rat. Studies of the kinetic parameters of the reverse reaction catalyzed by UDP-glucuronyltransferase indicate that the enzyme from Gunn rats also has decreased affinity for UDP. Calculated values of deltaG degrees for the binding of the UDP portion of UDP-glucuronic acid suggest that the defect of UDP-glucuronyltransferase of Gunn rats appears limited to abnormal interactions between the enzyme and the UDP portion of UDP-glucuronic acid. Studies of the extent of UDP-induced inhibition of the forward reaction support this idea. Diethylnitrosamine, added to microsomes in vitro, enhances the affinity of UDP-glucuronyltransferase for the UDP portion of UDP-glucuronic acid. Despite the defective conformation of the UDP-glucuronic acid binding site of UDP-glucuronyltransferase from Gunn rats this enzyme is activated in the normal way by UDP-N-acetylglucosamine, which is a K-type effector with regard to UDP-glucuronic acid.

Allosteric Regulation↗
PubMed2002

Core protein dependence of epimerization of glucuronosyl residues in galactosaminoglycans.

Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl and l-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-S353), with two glycosaminoglycan chains, also contained almost exclusively d-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3'-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with approximately 15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.

Amino Acid Sequence↗
PubMed1974

Inducibility of beta-glucuronidase in wild-type and hexuronate-negative mutants of Escherichia coli K-12.

In strain K-12 of Escherichia coli, beta-glucuronidase synthesis was induced only by beta-glucuronides: all intermediates of the hexuronate pathway able to enter the cells failed to induce the enzyme significantly. The induction pattern of beta-glucuronidase clearly differentiates the mode of regulation of its synthesis from those of the subsequent enzymes of the pathway, which are induced by fructuronate and/or tagaturonate. In mutant strains blocked in glucuronate metabolism after the isomerase step, beta-glucuronidase synthesis was still induced by a beta-glucuronide. Glucuronate and fructuronate, which are accumulated and mutually interconverted within the cells, become good inducers of beta-glucuronidase: they induce up to a level one-half that obtained in the wild-type strain in the presence of beta-glucuronide alone. In an isomerase-negative strain where fructuronate is not produced, beta-glucuronidase was no longer induced by beta-glucuronide unless supplemented with fructuronate. In this strain, glucuronate alone or fructuronate alone exhibited greater inducing ability than in the wild-type strain. Moreover, fructuronate could also enhance glucuronate-induced synthesis of beta-glucuronidase. Glucuronate was not able to activate beta-glucuronideinduced synthesis of beta-glucuronidase. Therefore, the induction of beta-glucuronidase synthesis depends upon two factors which, when acting separately, are both poor inducers but can act cooperatively; one factor is beta-glucuronide or glucuronate and the second is fructuronate. The specific inducing capacity of each of these three compounds as well as the hypothetical mechanism(s) of the action of fructuronate are discussed.

Alcohol Oxidoreductases↗
PubMed1991

Internal alkalinization by reversal of anion exchange in human neutrophils: regulation of transport by pH.

When steady-state human neutrophils bathed in 148 mM Cl- are transferred to a Cl(-)-free medium containing 0.5 mM HCO3- and 148 mM glucuronate or aspartate as nominally inert replacement ions, there is a rapid efflux of 36Cl- from the cells. The accelerated loss of Cl- is accompanied by an intracellular alkalinization of 0.7-0.9 pH units. Both the Cl- efflux and intracellular pH (pHi) transient are dependent on extracellular HCO3- and are sensitive to inhibition by SITS and alpha-cyano-4-hydroxycinnamate, which block anion exchange, thereby indicating that these processes are due to the countertransport of internal Cl- for external HCO3-. Rate of anion exchange is strongly influenced by pH, which functions to regulate carrier activity; alkalinization stimulates the transport velocity, whereas acidification inhibits it. The relationship to pHi follows a Hill equation with pK' approximately 7.40 and Hill coefficient of 3.3, thereby suggesting that approximately 3 HCO3- may be required to bind to the modifier site. Neutrophils placed in glucuronate medium progressively shrink during the first 7.5 min of incubation due to the net loss of osmotically active particles through Cl(-)-HCO3- exchange. However, between 7.5 and 30 min, cells regain their normal cell size. This volume recovery phase correlates with the time course of 22Na+ and [14C]glucuronate influxes, whose kinetics can be dissociated from that of anion exchange. Uptake of glucuronate is largely Na+ dependent (whereas Cl(-)-HCO3- exchange is not), is resistant to amiloride, and can be blocked by furosemide, which suggests that glucuronate probably enters via a volume-activated pathway such as Na+ + glucuronate cotransport.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1983

Modulation of the number of ligand binding sites of UDP-glucuronyltransferase by the gel to liquid-crystal phase transition of phosphatidylcholines.

The kinetics of a pure, delipidated form of microsomal UDP-glucuronyltransferase is non-Michaelis-Menten when the enzyme is reconstituted into unilamellar vesicles of phosphatidylcholine that are in a gel phase. Double reciprocal plots of velocity as a function of the concentration of UDP-glucuronic acid show a downward curvature under these conditions. Binding studies indicate that the basis for the kinetic pattern is the presence of one high affinity and one low affinity binding site for UDP-glucuronic acid. The two classes of binding sites seem to be generated by the presence of two subunits that bind UDP-glucuronic acid within a single molecule of UDP-glucuronyltransferase. Melting the phospholipids from the gel phase to the liquid-crystal phase is associated with a switch from non-Michaelis-Menten to Michaelis-Menten kinetics for UDP-glucuronyltransferase. Binding studies for interaction of UDP-glucuronic acid with enzyme present in a liquid-crystal lipid phase indicate that the two binding sites for UDP-glucuronic acid do not become identical in this setting. Instead, one of the sites becomes nonfunctional. Binding studies carried out with UDP as ligand lead to similar results. There is a high affinity and a low affinity site for UDP when enzyme is reconstituted into a phospholipid bilayer in a gel phase. There is only one UDP binding site per holoenzyme when enzyme is reconstituted into a phospholipid bilayer in a liquid-crystal phase. Delipidated enzyme or enzyme reconstituted with lysophosphatidylcholine displays Michaelis-Menten kinetics. Binding studies show that these forms have only one binding site for UDP-glucuronic acid per holoenzyme. However, they have two nonidentical binding sites for binding of UDP. Thus, the physical properties of its phospholipid milieu influence the number of functional binding sites of UDP-glucuronyltransferase.

Animals↗
PubMed1996

Reproductive steroids during maturation in a primitive teleost, the Pacific herring (Clupea harengus pallasi).

Concentrations of reproductive steroids were measured in the plasma of captive Pacific herring, Clupea harengus pallasi, (1) prior to ovulation and milt production, (2) during the periovulatory and newly milt-producing period, (3) during a "ripe" holding period after ovulation and during milt production, and (4) before and after spawning. 17alpha,20beta-Dihydroxyprogesterone (17,20beta-P), despite being present only in low concentrations in the unconjugated (free) form (<10 ng/ml), is likely to be the maturation inducing steroid in females and was associated with the initiation of milt production in males since its levels are elevated coincidentally with these events. Glucuronated 17,20beta-P, free 17alpha-hydroxyprogesterone (17-P), and free and glucuronated 3alpha, 17alpha-dihydroxy-5beta-pregnan-20-one (3alpha,17-P-5beta) were present in high concentrations (140-250 ng/ml) in periovulatory females and newly milt-producing males. This steroid pattern suggests that the low levels of 17,20beta-P are due to glucuronation and competitive conversion of its precursor, 17-P, to free and glucuronated 3alpha, 17-P-5beta. Glucuronated testosterone was the principal steroid in preovulatory and premilt-producing fish (200-350 ng/ml), coincident with similar levels of glucuronated 11-ketotestosterone in males. After ovulation females did not spawn synchronously until 2 months later, which may be partially due to reduced environmental cues in the captive situation, while male fish released milt sporadically throughout the ripe holding period. Steroidal indicators of readiness to spawn in females or males were not detected. Rather, levels of all steroids gradually decreased in ripe holding fish (<30 ng/ml) to reach even lower levels (<1 ng/ml) after spawning. We suggest that "runniness" of gametes is a distinctive characteristic of females that are ready to spawn, but that this may result from relaxation of sphincter muscles rather than being an additional maturational step.

17-alpha-Hydroxyprogesterone↗
PubMed1976

Investigations on the kinetic properties of estrone glucuronyltransferase from pig kidney.

The microsomal fraction of the pig kidney catalyzes the glucuronidation of estrone in the presence of UDP-glucuronic acid. This bireactant system exhibits a sequential type of reaction mechanism. Increasing concentrations of either substrate increase the affinity of the enzyme for the other substrate. The Hill coefficient, n, was calculated to be 1.0 for both estrone and UDP-glucuronic acid. The Kestrone and KUDP-glucuronic acid are 6.6 muM and 254 muM, respectively. The estrone glucuronyltransferase (UDP-glucuronate: 17 beta-oestradiol 3-glucuronosyltransferase, EC 2.4.1.59) exhibits high substrate specificity in that it is inhibited noncompetetively by estradiol-17 beta, estradiol-17 alpha, estriol, testosterone, phenolphthalein and bilirubin; p-nitrophenol and o-aminophenol do not inhibit the glucuronidation of estrone. Mg2+ and Ca2+ were found to be nonessential activators. One of the two products of the reaction, estrone glucuronide, inhibits the enzyme competitively in the presence of increasing concentrations of UDP-glucuronic acid. The other product of the reaction, UDP, inhibits the enzyme noncompetitively with varying estrone concentrations and uncompetitively with varying UDP-glucuronic acid concentrations. Under incubation conditions for the glucuronidation of estrone, the enzyme catalyzes the reverse reaction with estrone glucuronide and UDP as reactants to an extent of about 0.4% of the forward reaction; this reverse reaction is also of a sequential type.

Adenosine Triphosphate↗
PubMed2006

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.

Aldose-Ketose Isomerases↗