scholarly journals Sequential degradation of a chondroitin sulphate trisaccharide by lysosomal enzymes from embryonic-chick epiphysial cartilage

1979 ◽  
Vol 179 (1) ◽  
pp. 7-13 ◽  
Author(s):  
B Ingmar ◽  
A Wasteson
Keyword(s):  
Lysosomal Enzymes ◽  
Glucuronic Acid ◽  
Enzyme Inhibitors ◽  
Functional Unit ◽  
Chondroitin Sulphate ◽  
Cartilage Degradation ◽  
Ester Group ◽  
Embryonic Chick ◽  
Reaction Products ◽  
Epiphysial Cartilage

The disulphated trisaccharide D-N-acetylgalactosamine sulphate-beta-D-glucuronic acid-beta-D-N-acetylgalactosamine sulphate prepared from 35S- or 14C-labelled chondroitin sulphate was incubated with a preparation of lysosomal enzymes from embryonic-chick epiphysial cartilage. Degradation was demonstrated by analysis of the reaction products. By use of the appropriate intermediate products as substrates, in conjunction with specific enzyme inhibitors, it was shown that the degradation proceeded sequentially from the non-reducing end. It was initiated by sulphatase (preferentially hydrolysing sulphate ester groups at the 6-position), followed by beta-N-acetylgalactosaminidase and beta-glucuronidase, converting the substrate into monosaccharides and inorganic sulphate. The latter enzyme preferentially attacked disaccharides carrying their sulphate ester group at C-4 of the hexosamine residue. Generation of chondroitin sulphate oligosaccharides may occur by the action of an endoglycosidase, previously demonstrated in embryonic-chick cartilage. Endo- and exo-enzymes may thus form a functional unit in lysosomal degradation of chondroitin sulphate.

scholarly journals Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals α/β Hydrolase Folds for d-β-Acetylthioisobutyric Acid Synthesis

2005 ◽  
Vol 187 (24) ◽  
pp. 8470-8476 ◽  
Author(s):  
Fatemeh Elmi ◽  
Hsin-Tai Lee ◽  
Jen-Yeng Huang ◽  
Yin-Cheng Hsieh ◽  
Yu-Ling Wang ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Enzyme Inhibitors ◽  
Specific Activity ◽  
Methyl Esters ◽  
Enantiomeric Excess ◽  
Angiotensin Converting Enzyme Inhibitors ◽  
Acid Synthesis ◽  
Ester Group ◽  
Catalytic Triad ◽  
Converting Enzyme Inhibitors

ABSTRACT Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (dl-MATI) to produce d-β-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 Å, confirming that EST is a member of the α/β hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ∼12 Å deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

scholarly journals Biodiversity of CS–proteoglycan sulphation motifs: chemical messenger recognition modules with roles in information transfer, control of cellular behaviour and tissue morphogenesis

2018 ◽  
Vol 475 (3) ◽  
pp. 587-620 ◽  
Author(s):  
Anthony Hayes ◽  
Kazuyuki Sugahara ◽  
Brooke Farrugia ◽  
John M. Whitelock ◽  
Bruce Caterson ◽  
...  
Keyword(s):  
Information Transfer ◽  
Enzyme Inhibitors ◽  
Chondroitin Sulphate ◽  
Chain Structure ◽  
Bioactive Molecules ◽  
Biological Information ◽  
Extracellular Milieu ◽  
Overwhelming Evidence ◽  
Chemical Messenger ◽  
Cellular Behaviour

Chondroitin sulphate (CS) glycosaminoglycan chains on cell and extracellular matrix proteoglycans (PGs) can no longer be regarded as merely hydrodynamic space fillers. Overwhelming evidence over recent years indicates that sulphation motif sequences within the CS chain structure are a source of significant biological information to cells and their surrounding environment. CS sulphation motifs have been shown to interact with a wide variety of bioactive molecules, e.g. cytokines, growth factors, chemokines, morphogenetic proteins, enzymes and enzyme inhibitors, as well as structural components within the extracellular milieu. They are therefore capable of modulating a panoply of signalling pathways, thus controlling diverse cellular behaviours including proliferation, differentiation, migration and matrix synthesis. Consequently, through these motifs, CS PGs play significant roles in the maintenance of tissue homeostasis, morphogenesis, development, growth and disease. Here, we review (i) the biodiversity of CS PGs and their sulphation motif sequences and (ii) the current understanding of the signalling roles they play in regulating cellular behaviour during tissue development, growth, disease and repair.

Histochemical Differentiation of the Basement Membrane of the Mouse Seminiferous Tubule

1962 ◽  
Vol s3-103 (63) ◽  
pp. 385-391
Author(s):  
A. H. BAILLIE
Keyword(s):  
Basement Membrane ◽  
Protein Metabolism ◽  
Seminiferous Tubule ◽  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Hair Follicles ◽  
Ground Substance ◽  
Seminiferous Tubules ◽  
Active Protein ◽  
Pas Reaction

The ground substance of the testis of the albino mouse is PAS-positive but not metachromatic, and probably highly aggregated. The basement of the seminiferous tubules is intensely PAS-positive, metachromatic, and possibly not so highly aggregated. The reactivity of the ground substance to the PAS reaction and toluidine blue is tentatively ascribed to the presence of chondroitin sulphate C: this compound, previously known to contain N acetyl-galactosamine, glucuronic acid, tyrosine and tryptophane, is associated with arginine. The genesis of the basement membrane of the seminiferous tubule is shown to include the formation of a sheath of atypical elongated fibroblasts, the secretion of a PAS positive, metachromatic substance associated with arginine between this sheath and the seminiferous tubule, the appearance of mitochondria in the cells of the sheath, and lastly, the acquisition of alkaline phosphatase by these fibroblasts and its spread to the intervening ground substance. These changes are thought to be related to the structural and nutritional requirements of the seminiferous tubules. In its intense positive reaction to PAS and in its metachromasy, the basement membrane of the seminiferous tubule agrees with the ground substance adjacent to sites of active protein metabolism, such as growing tumours, embryonic organs, hair follicles, and skin.

scholarly journals Inhibition of leucocytic lysosomal enzymes by glycosaminoglycans in vitro

1975 ◽  
Vol 152 (1) ◽  
pp. 57-64 ◽  
Author(s):  
J L Avila ◽  
J Convit
Keyword(s):  
Incubation Medium ◽  
Lysosomal Enzymes ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Polymorphonuclear Leucocyte ◽  
Dependent Manner ◽  
Acid Hydrolases ◽  
Lysosomal Fraction ◽  
Formation Of Complexes

1. A lysosomal fraction was separated by density-gradient centrifugation from a highly purified human polymorphonuclear leucocyte suspension. 2. Some 23 different lysosomal enzymes were assayed for activity in the presence of various concentrations of glycosaminoglycans. 3. The 21 acid hydrolases assayed were strongly inhibited to different degrees by low (0-12 mmol/l) concentrations of glycosaminoglycans in a pH-dependent manner. Thus inhibitions were stronger below pH4.5, with activity returning to control values at about pH5.0. 4. On a molar basis, the inhibitory activity for the several glycosaminoglycans studied was: heparin > chondroitin sulphate ≫ hyaluronic acid. 5. Once the glycosaminoglycan-acid hydrolase complex was formed, it was partially dissociated by slight elevations in the pH of the incubation medium, by increasing the ionic strength of the incubation medium, or by adding several cationic proteins (e.g. histone, protamine). 6. As leucocytic lysosomes contain large amounts of chondroitin sulphate, and have a strongly acid intragranular pH, we suggest that glycosaminoglycans may modify lysosomal function through the formation of complexes with lysosomal enzymes, by inhibiting the digestive activity of the acid hydrolases when the intralysosomal pH is below their pI.

Chondroitin - application in the treatment of degenerative joint disease

2016 ◽  
Vol 18 (6) ◽  
pp. 621-628 ◽  
Author(s):  
Wiesław Tomaszewski
Keyword(s):  
Molecular Level ◽  
Proteolytic Enzymes ◽  
Chondroitin Sulphate ◽  
Cartilage Degradation ◽  
Degenerative Joint Disease ◽  
Joint Disease ◽  
Chondrocyte Apoptosis ◽  
Structural Elements ◽  
Acid Biosynthesis ◽  
Physiological Processes

Chondroitin is an organic compound, belonging to the group of glycosaminoglycans. In the treatment of degenerative joint disease, aka osteoarthritis, chondroitin sulphate is applied as a medicine or a dietary supplement. The biological importance of chondroitin sulphate has been already largely determined. The newest data on glycobiology research suggest that proteoglycans, as well as their complex polysaccharide macroparticles not only are the structural elements, but also they participate in multiple metabolic processes at a molecular level as well as in the physiological processes, regulating this type of mechanisms. The preparations applied in the treatment of degenerative joint disease, containing chondroitin sulphate, are attributed numerous therapeutic and chondroprotective properties including stabilizing synthesis processes and cartilage degradation through stimulation and inhibition of chondrocyte apoptosis (production of the elements of the intracellular substance and osteocyte stimulation), an increased proteoglycan and hyaluronic acid biosynthesis, inhibition of the activity of proteolytic enzymes and hyaluronidase, reduction of inflammatory mediators (prostaglandins and leukotrienes) and a decreased collagen II degradation. Based on the results of the multidirectional research available in the newest source literature, the analysis of the therapeutic efficacy and safety of chondroitin application in the treatment of degenerative joint disease was conducted.

scholarly journals Hydrazinolysis of heparin and other glycosaminoglycans

1984 ◽  
Vol 217 (1) ◽  
pp. 187-197 ◽  
Author(s):  
P N Shaklee ◽  
H E Conrad
Keyword(s):  
Chemical Modification ◽  
Carboxy Group ◽  
Uronic Acid ◽  
Glucuronic Acid ◽  
Chondroitin Sulphate ◽  
Acid Hydrazide ◽  
Iduronic Acid ◽  
Heparin Treatment ◽  
Hydrazine Sulphate ◽  
Low Rate

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.

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 Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta

1986 ◽  
Vol 240 (2) ◽  
pp. 575-583 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
T N Wight
Keyword(s):  
Uronic Acid ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polymer Concentration ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Structural Units ◽  
The Core ◽  
Covalently Linked

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 × 10(6) (PG-25), 0.30 × 10(6) (PG-35) and 0.88 × 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

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