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Sulfomucopolysaccharide or acid mucoprotein with sulfate group is detected in mantle mucus of some bivalves4-7), and is considered to act as a calcium carrier and provide calcium concentration high enough for the growth of shell minerals at the site of mineralization4). Histochemical and biochemical studies have suggested that the inner and outer mucus of the mantle of Pinctada fucata show metachromasia on staining with toluidine blue and are very similar to each other in their amino acid compositions1). If the acid mucous substances play the above-mentioned functional role in the calcium metabolism of molluscs, the mucus does not always form a shell. To answer this quesion, biochemical analysis of the acid polysaccharide was made on the inner and outer mucus of the mantle of Pinctada fucata and Chlamys nobilis. Inner mucus adhered on the inner surface of the mantle and outer mucus secreted between the mantle and shell were collected with an injector. In each case the dry mucus obtained by addition of ethyl alcohol was digested with “pronase” at pH 8.0 and 50°C for 72 hours and subsequently with trypsin at pH 7.5 and 37°C for 72 hours. Acid polysaccharide was then separated by the cetyl pyridinium chloride treatment and analysed as described in the preceding paper2). The amount and chemical composition of fraction A containing acid mucopolysaccharide are shown in Tables 1 and 2. The fraction contains a relatively large amount of sulfuric ester and a little amount of protein, but not chondroitine sulfate, as shown by paper electrophoresis. Galactosamine, glucosamine, galactose and glucose are detected in fraction A of both the inner and outer mantle mucus of Pinctada fucata by paper chromatography (Table 3). The fraction seems to be composed of sulfomucopolysaccharide or sulfated polysaccharide-protein complex. From these results, it may be concluded that the presence of the acid mucous substance with sulfate group is necessary to induce shell mineralization though it does not always induce it.

By means of enzymatic digestion with chondroitinase-ABC, a novel unsaturated disaccharide bearing two sulfate residues has been derived from chondroitin sulfate of squid cartilage. Various data, particularly the formation of 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose and its 6-O-sulfate isomer by hydrolysis with chondro-6-sulfatase and chondro-4-sulfatase, respectively, and the formation of 2-acetamido-2-deoxy-4,6-di-O-sulfo-d-galactose by mild acid hydrolysis, indicate that the two sulfate residues are located at positions 4 and 6 of the hexosamine moiety. A distinct unsaturated disaccharide also bearing two sulfate residues has been obtained from chondroitin sulfate B preparations of bovine lung and pig skin. One of the sulfate residues has been shown to be substituted at position 4 of the hexosamine moiety. The resistance of the second residue to both chondro-4-sulfatase and chondro-6-sulfatase and of the uronic acid moiety to Flavobacterium heparinum glucuronidase suggests that the sulfate residue is substituted at position 2 or 3 of the uronic acid moiety. These disulfated disaccharides are, therefore, isomers of 2-acetamido-2-deoxy-3-O-(2- or 3-O-sulfo-β-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose, the compound previously found in the digest of chondroitin sulfate from shark cartilage. The three isomeric disulfated disaccharides were separated from nonsulfated and monosulfated homologues and from one another by paper chromatography in 1-butyric acid-0.5 n ammonia (5:3). The separation of disaccharides from chondroitinase digests by paper chromatography permits comparison of chondroitinase digests from different chondroitin sulfate preparations and detection of slight variations in the types of sulfate linkage.

1. 1. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nosulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractioned by NaCl elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.