scholarly journals Initiation of chondroitin sulphate synthesis by β-d-galactosides. Substrates for galactosyltransferase II

1985 ◽  
Vol 227 (3) ◽  
pp. 805-814 ◽  
Author(s):  
J A Robinson ◽  
H C Robinson
Keyword(s):  
Chick Embryo ◽  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Acetate Incorporation ◽  
Glycosidic Linkage ◽  
Microsomal Preparation ◽  
Galactose Residue ◽  
Chain Synthesis ◽  
Chondroitin Sulphate Chain

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.

scholarly journals Control of chondroitin sulphate biosynthesis. β-d-Xylopyranosides as substrates for UDP-galactose: d-xylose transferase from embryonic-chicken cartilage

1981 ◽  
Vol 194 (3) ◽  
pp. 839-846 ◽  
Author(s):  
J A Robinson ◽  
H C Robinson
Keyword(s):  
Transfer Reaction ◽  
Chondroitin Sulphate ◽  
Acetate Incorporation ◽  
Microsomal Preparation ◽  
Embryonic Chicken ◽  
A Cell ◽  
Embryonic Cartilage ◽  
Chondroitin Sulphate Chain ◽  
Core Production

Embryonic-chicken epiphyseal cartilage was incubated in vitro with a variety of beta-xylosides and the amount of [3H]acetate incorporation into chondroitin sulphate was determined under conditions when normal protein core production was inhibited by cycloheximide. The ability of the different beta-xylosides to relieve thea cycloheximide-mediated inhibition of chondroitin sulphate synthesis was influenced by the nature of the aglycan group of te xyloside. beta-Xylosides with apolar and uncharged aglycan groups were most effective and produced a severalfold stimulation of chondroitin sulphate biosynthesis. beta-Xylosides with charged aglycan groups were less effective initiators of chondroitin sulphate synthesis. The rate of galactose transfer from UDP-galactose to each of the beta-xylosides, catalysed by a cell-free microsomal preparation from embryonic cartilage, was measured. This study showed that the nature of the aglycan group of the beta-xyloside was a factor determining the capacity of the xyloside to act as an acceptor for galactosyltransferase I, the enzyme that catalyses the first galactose transfer reaction of chondroitin sulphate synthesis. The aglycan group of the xyloside also appeared to influence other steps leading to chondroitin sulphate chain initiation in vitro.

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.

scholarly journals Incorporation of sulphate by chick-embryo corium. Nature and products of the process in vitro

1965 ◽  
Vol 97 (2) ◽  
pp. 432-439
Author(s):  
NL Noble ◽  
RJ Boucek
Keyword(s):  
Mast Cells ◽  
Chick Embryo ◽  
Phosphate Buffer ◽  
Trichloroacetic Acid ◽  
Chondroitin Sulphate ◽  
Energy Requirement ◽  
Heparan Sulphate ◽  
Energy Of Activation ◽  
Sulphate Concentration

1. The incorporation of sulphate into the trichloroacetic acid-precipitable fraction of 9-day chick-embryo corium, incubated in Krebs-Ringer phosphate buffer, pH7, is dependent on the sulphate concentration of the medium. Uptake of sulphate is linear with time for 3.5-4hr. and is maximal at 37.5 degrees in the presence of air or oxygen. d-Glucose stimulates the incorporation of sulphate but l-glutamine has no effect. 2. Incorporation of sulphate by the chick corium is enzymic and apparently involves the synthesis of active sulphate (adenosine 3‣-phosphate 5‣-sulphatophosphate) and the transfer of sulphate from adenosine 3‣-phosphate 5‣-sulphatophosphate to acceptor glycosaminoglycuronoglycan. This proposal on the nature of the process is suggested by the similarity between the energy of activation calculated for sulphate-incorporation in the chick-corium preparation and the energy requirement reported for sulphate-activation with purified yeast enzymes. 3. The 9-day chick-embryo corium is composed principally of fibroblasts; there are no histologically demonstrable mast cells. The young fibroblast is apparently responsible for the incorporation of sulphate into glycosaminoglycuronoglycans tentatively identified as chondroitin sulphate(s), heparan sulphate and heparin-like material.

scholarly journals Bilirubin glucuronyltransferase. Specific assay and kinetic studies

1971 ◽  
Vol 125 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Kim Ping Wong
Keyword(s):  
Glucuronic Acid ◽  
Helix Pomatia ◽  
Diazo Compound ◽  
Major Product ◽  
Disodium Salt ◽  
Final Concentration ◽  
Transferase Activity ◽  
Microsomal Preparation ◽  
Specific Assay

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg2+ and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-14C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with β-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ‘equivalents’ conjugated/h per mg of protein and 16.9–18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The Km values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3×10−4m and 1.67×10−3m, respectively. 7. The addition of Mg2+ at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1–20mm has no effect on the glucuronidation of bilirubin in vitro.

scholarly journals The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage

1984 ◽  
Vol 224 (3) ◽  
pp. 977-988 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Bovine Articular Cartilage ◽  
The Core ◽  
Elevated Rate ◽  
Chain Synthesis ◽  
Chondroitin Sulphate Chain ◽  
Bovine Cartilage

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.

scholarly journals The effect of D-xylose, β-D-xylosides and β-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage

1975 ◽  
Vol 148 (1) ◽  
pp. 25-34 ◽  
Author(s):  
H C Robinson ◽  
M J Brett ◽  
P J Tralaggan ◽  
D A Lowther ◽  
M Okayama
Keyword(s):  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Polysaccharide Chain ◽  
Embryonic Chicken ◽  
Chain Synthesis

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, xβ-D-xylosides and β-D-galactosides to the incubation medium. β-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, β-D-xylosides and β-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.

scholarly journals Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity

1985 ◽  
Vol 226 (3) ◽  
pp. 705-714 ◽  
Author(s):  
M W Gundlach ◽  
H E Conrad
Keyword(s):  
Hyaluronic Acid ◽  
Chick Embryo ◽  
Chondroitin Sulphate ◽  
Chain Elongation ◽  
Optimal Conditions ◽  
Ph Optimum ◽  
Microsomal Preparation ◽  
Glycosyl Transferases

The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo.

Sign in / Sign up

Export Citation Format

Share Document