Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.
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PMID:A method for the sequence analysis of dermatan sulphate. 216 67

Aggrecan-derived chondroitin sulfate (CS) chains, released by beta-elimination, were derivatized with p-aminobenzoic acid or p-aminophenol; radioiodinated; and subjected to graded or complete degradations by chondroitin ABC lyase to generate linkage region fragments of the basic structure DeltaGlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (where DeltaGlyUA represents 4, 5-unsaturated glycuronic acid, and R is the adduct), by chondroitin AC lyase to generate the shorter fragment DeltaGlyUA-Gal-Gal-Xyl-R, or by chondroitin C lyase to generate the same fragment when it was linked to a 6-O-sulfated or unsulfated GalNAc at the nonreducing end. Fragments were separated by size using gel chromatography, by charge using ion-exchange chromatography, and by size/charge using electrophoresis and then characterized by stepwise degradations from the nonreducing end by using mercuric acetate to remove all terminal DeltaGlyUA, by bacterial glycuronidase to remove the same residue when linked to unsulfated or 6-O-sulfated GalNAc/Gal, by mammalian 4-sulfatase to remove sulfate from terminal GalNAc 4-O-sulfate, by chondro-4-sulfatase to remove 4-O-sulfate from other GalNAc/Gal residues, and by beta-galactosidase to remove terminal Gal. Results with CS from bovine nasal cartilage aggrecan show that, in nearly all chains, Xyl and probably also the first Gal are unsubstituted, whereas the second Gal is 4-O-sulfated in one CS chain out of five. The first disaccharide repeat is sulfated at C-4 of GalNAc in one chain out of three and unsulfated in the other two. A sulfated first disaccharide is always joined to an unsulfated GlcUA-Gal-Gal sequence. In contrast, CS from human articular cartilage usually has a sulfated first disaccharide repeat. In CS from young human cartilage, sulfate groups are mostly at C-4 of GalNAc in the major part of the chain, but at C-6 in the nonreducing distal portion. In CS from old cartilage, sulfation at C-6 of GalNAc is a major feature from the nonreducing end down to approximately positions 4 and 5 from the linkage region, where GalNAc 4-O-sulfate is common.
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PMID:Variations in the chondroitin sulfate-protein linkage region of aggrecans from bovine nasal and human articular cartilages. 891 Apr 87