scholarly journals Biosynthesis of glycosaminoglycans in bovine cornea. The effect of uridine diphosphate xylose

1970 ◽  
Vol 120 (4) ◽  
pp. 719-723 ◽  
Author(s):  
C. Balduini ◽  
A. Brovelli ◽  
A. A. Castellani
Keyword(s):  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Glucose Dehydrogenase ◽  
Uridine Diphosphate ◽  
Regulatory Effect ◽  
Keratan Sulphate ◽  
Polysaccharide Chain

1. The role of UDP-xylose in the regulation of corneal glycosaminoglycan biosynthesis was investigated. Bovine corneas were incubated with [U-14C]-glucose in the presence and in the absence of the nucleotide, and the radioactivity of chondroitin, chondroitin sulphate and keratan sulphate, as well as of their monosaccharide constituents, was determined. 2. A decrease in the rate of biosynthesis of chondroitin and chondroitin sulphate and an increase in that of keratan sulphate were observed in the samples incubated with UDP-xylose. 3. The UDP-glucuronic acid isolated after the incubation in the presence of UDP-xylose showed a noticeable decrease in the amount of radioactivity incorporated; this result suggests that UDP-xylose inhibits the UDP-glucose dehydrogenase, causing an accumulation of UDP-glucose and consequently an increase in the formation of UDP-galactose and keratan sulphate. 4. Galactose and galactosamine isolated from the polysaccharides showed variations in the amount of radioactivity incorporated in accordance with those observed for the macromolecules; this fact confirms that in the system we used in vitro a real biosynthesis of the polysaccharide chain took place and that the regulatory effect of UDP-xylose was active at the monosaccharide level.

The extracellular matrix of the developing cornea: diversity, deposition and function*

Development ◽  
1988 ◽  
Vol 103 (Supplement) ◽  
pp. 195-205
Author(s):  
J. B. L. Bard ◽  
M. K. Bansal ◽  
A. S. A. Ross
Keyword(s):  
Extracellular Matrix ◽  
Chondroitin Sulphate ◽  
Corneal Stroma ◽  
Experimental System ◽  
Collagen I ◽  
And Function ◽  
20 Nm

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.

A DFT study on the catalytic mechanism of UDP-glucose dehydrogenase

2010 ◽  
Vol 88 (8) ◽  
pp. 804-814 ◽  
Author(s):  
WenJuan Huang ◽  
Jorge Llano ◽  
James W. Gauld
Keyword(s):  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Polarizable Continuum Model ◽  
Uridine Diphosphate ◽  
Reaction Field ◽  
Enzyme Active Site ◽  
Site Model ◽  
Diphosphate Glucose

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

THE ACID-SOLUBLE NUCLEOTIDES OF SALMON LIVER

1958 ◽  
Vol 36 (5) ◽  
pp. 465-473 ◽  
Author(s):  
H. Tsuyuki ◽  
Violet M. Chang ◽  
D. R. Idler
Keyword(s):  
Low Temperature ◽  
Anion Exchange ◽  
Rat Liver ◽  
Glucuronic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Uridine Diphosphate ◽  
Carbohydrate Storage ◽  
Diphosphate Glucose

The acid-soluble nucleotides of spring salmon liver have been separated by anion-exchange chromatography at low temperature and characterized. Under these conditions the relatively labile uridine-5′-diphosphate nucleotides of acetylglucosamine, galactose, and glucuronic acid were obtained intact, a fact that is further substantiated by the complete absence of uridine-5′-diphosphate. The occurrence of these uridine diphosphate compounds and the absence of uridine diphosphate glucose is discussed in relation to the previously postulated role of inositol as a carbohydrate storage product. A new peptide-containing nucleotide, succinoadenosine-5′-phosphosulphate (peptide), was found in the fraction which immediately follows adenosine-5′-diphosphate. The parent base of this nucleotide, succinoadenine, was also isolated. The nucleotide pattern is simpler than that reported by other investigators for rat liver and wheat.

scholarly journals The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

1974 ◽  
Vol 141 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Paul A. Gainey ◽  
Charles F. Phelps
Keyword(s):  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Protein Fluorescence ◽  
Diphosphate Glucose ◽  
Ph Dependent ◽  
Uridine Diphosphate Glucose Dehydrogenase ◽  
Biphasic Profile

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.

The Role of the Microsomal Membrane in Control of Uridine Diphosphate Glucuronyltransferase Activity in vitro and in vivo

1974 ◽  
Vol 2 (6) ◽  
pp. 1167-1172 ◽  
Author(s):  
A. B. GRAHAM ◽  
D. T. PECHEY ◽  
G. C. WOOD ◽  
B. G. WOODCOCK
Keyword(s):  
Microsomal Membrane ◽  
Uridine Diphosphate

scholarly journals Uridine diphosphate glucuronic acid production and utilization in various tissues actively synthesizing glycosaminoglycans

1972 ◽  
Vol 128 (2) ◽  
pp. 215-227 ◽  
Author(s):  
P. A. Gainey ◽  
C. F. Phelps
Keyword(s):  
Nasal Septum ◽  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Neonatal Rat ◽  
Uridine Diphosphate ◽  
Ph Profile ◽  
Nasal Cartilage ◽  
Space Experiments ◽  
Tissue Space ◽  
Wet Weight

1. UDP-glucose dehydrogenase has been partially purified from sheep nasal septum cartilage, neonatal rat skin and bovine corneal epithelium. 2. The pH profile, Km values for NAD+ and UDP-glucose, activation energy and molecular weight have been determined for the enzyme from several of the tissues. 3. The sugar nucleotide concentrations in each of the tissues have been related to the spectrum of glycosaminoglycans produced by each tissue. 4. The presence of an allosteric UDP-xylose-binding site distinct from the active site(s) in sheep nasal septum UDP-glucose dehydrogenase has been demonstrated. 5. An active UDP-glucuronic acid nucleotidase has been demonstrated in sheep nasal cartilage. 6. Tissue-space experiments have shown the cell water content of sheep nasal septum cartilage to be 14% of the wet weight. 7. Glucuronic acid 1-phosphate does not occur in measurable amounts in sheep nasal septum cartilage and no UDP-glucuronic acid pyrophosphorylase activity could be detected in this tissue. 8. The inhibition by UDP-xylose with respect to both substrates, UDP-glucose and NAD+, has been examined, and shown to be allosteric.

scholarly journals Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 2029-2039 ◽  
Author(s):  
Slade A. Loutet ◽  
S. Josefin Bartholdson ◽  
John R. W. Govan ◽  
Dominic J. Campopiano ◽  
Miguel A. Valvano
Keyword(s):  
Real Time Pcr ◽  
Biochemical Analysis ◽  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Polymyxin B ◽  
Burkholderia Cenocepacia ◽  
Pcr Analysis ◽  
Increased Sensitivity ◽  
In Trans

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugdBCAL2946 , ugdBCAM0855 and ugdBCAM2034 , all of which were individually inactivated. Only inactivation of ugdBCAL2946 resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugdBCAL2946 or ugdBCAM0855 but not ugdBCAM2034 was expressed from plasmids. The growth of a conditional ugdBCAL2946 mutant, created in the ΔugdBCAM0855 background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugdBCAL2946 or ugdBCAM0855 expressed in trans, but not by ugdBCAM2034 . Biochemical analysis of the purified, recombinant forms of UgdBCAL2946 and UgdBCAM0855 revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD+. Purified UgdBCAM2034 showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugdBCAL2946 was 5.4- and 135-fold greater than that of ugdBCAM0855 and ugdBCAM2034 , respectively. Together, these data indicate that the combined activity of UgdBCAL2946 and UgdBCAM0855 is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugdBCAL2946 , is required for polymyxin B resistance.

scholarly journals Structures of bilirubin conjugates synthesized in vitro from bilirubin and uridine diphosphate glucuronic acid, uridine diphosphate glucose or uridine diphosphate xylose by preparations from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 635-644 ◽  
Author(s):  
J. Fevery ◽  
P. Leroy ◽  
M. Van De Vijver ◽  
K. P. M. Heirwegh
Keyword(s):  
Carboxylic Acid ◽  
Rat Liver ◽  
Glucuronic Acid ◽  
Diazonium Salt ◽  
Acid Amide ◽  
Uridine Diphosphate ◽  
Prolonged Incubation ◽  
Carboxylic Acid Groups ◽  
Decreasing Ph

1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated: with (a), bilirubin β-d-monoglucuronoside, with (b), bilirubin β-d-monoglucoside and with (c), bilirubin monoxyloside or mixtures of the mono-and di-xyloside. 2. With UDP-glucuronic acid prolonged incubation and variation of the composition of the incubation mixtures yielded equimolar amounts of azodipyrrole (I) and azodipyrrole β-d-monoglucuronoside (II) after treatment of the incubation mixtures with the diazonium salt of ethyl anthranilate. The azo-derivatives were identified by t.l.c. by reference to known compounds and by the following chemical tests. After ammonolysis the conjugated azo-derivative (II) yielded d-glucuronic acid and the carboxylic acid amide of azodipyrrole, indicating transfer of a glucuronic acid residue to the carboxylic acid groups of bilirubin. The β-d-configuration of the sugar moiety and binding at C-1 were demonstrated by enzymic hydrolysis tests. 3. Analogous evidence established the structure of the reaction product obtained with UDP-glucose as the sugar donor, as bilirubin β-d-monoglucoside. 4. With UDP-xylose as the sugar donor xylosyl transfer to the carboxylic acid groups of bilirubin with attachment at C-1 was demonstrated in an analogous way. A β-d-configuration is considered very likely, but requires confirmation. 5. Monoxyloside formation was predominant at pH7.4, whereas at decreasing pH values increasing fractions of the substrate were converted into the dixyloside. Prolonged incubation, low concentrations of bilirubin and high concentrations of UDP-xylose favoured diconjugate formation. The available evidence supports the synthesis sequence: bilirubin → bilirubin monoxyloside → bilirubin dixyloside.

scholarly journals Chondroitin sulphate and keratan sulphate are almost isosteric

1991 ◽  
Vol 275 (1) ◽  
pp. 267-268 ◽  
Author(s):  
J E Scott
Keyword(s):  
Chondroitin Sulphate ◽  
Anionic Sites ◽  
Keratan Sulphate

Keratan sulphate and chondroitin sulphate (KS and CS) in the 2-fold helical configurations that are prevalent in solution are of very similar tacticity. The chiral centres, anionic sites and hydrophobic patches are in identical conformations. Only the position of the acetamido group varies from CS to KS, but part of its intramolecular H-bonding potential in CS is retained in KS. The formation of tertiary aggregates, observed in vitro and in tissues, is explicable on these bases. The proposal that KS may be a functional substitute for CS [Scott & Haigh (1988) J. Anat. 158, 95-108] under low-O2 conditions is relevant.

In Vitro Metabolism of 17β-Estradiol by Human Liver Tissue

1975 ◽  
Vol 53 (8) ◽  
pp. 903-906 ◽  
Author(s):  
R. Hobkirk ◽  
Joyce D. Mellor ◽  
Mona Nilsen
Keyword(s):  
Phosphate Buffer ◽  
Human Liver ◽  
Glucuronic Acid ◽  
Liver Homogenate ◽  
Uridine Diphosphate ◽  
Main Metabolite ◽  
Human Liver Tissue ◽  
17Β Estradiol ◽  
Human Liver Slices

17β-[6,7-3H]Estradiol was incubated with adult human liver slices in Krebs–Ringer phosphate buffer containing glucose. Of the identified 3H recovered, 51–76% consisted of estrone-3-sulfate (E13S) and 17β-estradiol-3-sulfate (E23S). E13S was the main metabolite and was found in both tissue and medium. E23S was present only in the medium. Minor amounts of estrogen glucuronides were formed. When a human liver homogenate was incubated with [3H]E2 in a medium fortified with excess uridine diphosphate glucuronic acid only some 4% of conjugation with glucuronic acid was observed. It is suggested that human liver favors sulfurylation as the conjugating mechanism for E2 and E1.

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