E.A. Davidson

Metabolism in vivo of connective-tissue mucopolysaccharides: I. Chondroitin sulfate C and keratosulfate of nucleus pulposus

Abstract The compositon of rabbit nucleus pulposus has been investigated as a function of age. The ratio of keratosulfate to chondroitin sulfate increases uniformly as a function of time. Several hormones, notably growth hormones, estrogens and androgens are able to alter the composition of mature tissues towards that represented by a younger age. The metabolic activity of the polysaccharides has been examined and their half-lives estimated. The keratosulfate of the mature animal appears to be extremely inert following synthesis and probably has a half-life in excess of 120 days.

Hexosamine and acid glycosaminoglycans in human teeth

1. 1. Human tooth dentine-cementum and enamel contain 0.08% and 0.03% hexosamine, respectively, with approximately equivalent amounts of glucosamine and galactosamine. 2. 2. Chondroitin 4-sulfate and/or chondroitin 6-sulfate are present in dentine-cementum and enamel of human teeth with smaller amounts of hyaluronic acid. There is apparently no dermatan or keratan sulfate. 3. 3. Chondroitin 6-sulfate is the major acid glycosaminoglycan in the dentine-cementum of human teeth.

UDP-d-glucuronic acid-5-epimerase and UDP-N-acetylglucosamine-4-epimerase of rabbit skin

Abstract UDP-GNAc-4-epimerase has been isolated from rabbit-skin extracts which had previously been found to contain UDP-GA-5-epimerase. Both enzymes fractionate similarly with ammonium sulphate, require catalytic NAD+ and are inhibited by NADH. NADP is not active in either case while thionicotinamide-AD+ and AP-AD+ are less active than NAD+. The inhibition of each epimerase by the others substrate as well as by UDPG, uracil, uridine, and UDP may be explained by a non-specific binding of the base and/or sugar moiety to the enzymes.

Isolation of anovel sulphatase from rat liver

Abstract A lysosomal sulphatase active towards an oligosaccharide sulphate, has been isolated from rat liver and partially purified. The purified system still contained β-glucuronidase, β-N-acetylhexosaminidase and arylsulphatase activities. The substrate employed for the assay was isolated from the digestion products obtained from chick embryo chondroitin sulphate A following testicular hyaluronidase digestion. Specificity studies indicated that the system was only active towards relatively high molecular weight substrates, but not towards chondroitin sulphate A itself. Prior cleavage of the nonterminal glucuronosyl residue of the oligosaccharide was apparently essential in the overall hydrolytic process.

Metabolism in vivo of connective-tissue mucopolysaccharides: III. Chondroitin sulfate and keratosulfate of cartilage

Abstract The incorporation of glucose into the hexosamine moieties of cartilage mucopolysaccharides has been studied. Isotope appears in the galactosamine moiety of the light mucoprotein fraction first and rapidly disappears from this fraction with an accompanying appearance of isotope in the insoluble residue. Chondroitin sulfate accounts for about 80% of the total polysaccharide and appears to be relatively inert metabolically. Appearance of label in chondroitin sulfate prior to keratosulfate suggests that these polysaccharides do not represent part of the same macromolecular structure. This latter point is not established. The effect of several hormones on the incorporation and turnover of isotope in this tissue was also reported.

Hexosamine and hydroxyproline alterations in chronically sun-damaged skin.

Summary 1. The upper dermis of chronically sun-damaged human skin contains more hexosamine and less hydroxyproline than the upper dermis from unexposed areas. 2. The increased hexosamine confirms the presence of increased acid mucopolysaccharides seen histologically in sun-damaged skin, but the decreased hydroxyproline does not identify the elastin staining material as either elastin or “degraded” collagen. 3. The upper dermis of sun-damaged skin has higher hexosamine and lower hydroxyproline levels than the lower-most dermis of the same specimen, thus the localization of the biochemical alteration corresponds with the localization of the histologic abnormality.

Ion-exchange separation and automated assay of some hexosamines: Part I

Abstract Separation of various 2-amino-2-deoxy- d -hexoses, 2-amino-2,6-dideoxy- d -hexoses, 2,6-diamino-2,6-dideoxy- d -hexoses, 3-amino-3-deoxy- d -hexoses, and 3-amino-3,6-dideoxy- d -hexoses, as well as 2-amino-2-deoxy- d -hexuronic acids and d -muramic acids, has been effected on Dowex 50 resins using pyridine-acetate buffers. The column effluents were assayed by reaction with ninhydrin in a Technicon AutoAnalyzer.

Studies on porcine costal cartillage protein-polysaccharide complex. I. Enzymatic degradation

Abstract Enzymatic degradation of porcine costal cartillage protein-polysaccharide complex with papain (EC 3.4.4.10) followed by fractionation on Sephadex and Sephadex ion-exchange columns yields two distinct polysaccharide fractions, designated A and B. The former, major fraction, still contains covalently-bound peptide and the majority of the glucosamine content of the parent polysaccharide. Hydrazinolysis experiments suggest that serine is the predominant, if not exclusive, residue to which the polysaccharide chains of this fraction are attached. The second polysaccharide fraction contains essentially no glucosamine, little or no polypeptide, and may be linked to the peptide in a different manner from the first fraction. Alkali elimination of polysaccharide from Fraction A followed by high-voltage electrophoresis yields several peptides; the partial structure of one of these was determined as Glu-Gly-dehydroAla-Gly. It is not possible to reconstruct the entire complex but this relatively unhindered linkage may be common for substitution of reactive serines with polysaccharide chains. Both xylose and galactose were identified in Fraction A by gas-liquid chromatography.

Disaccharidase of rabbit small intestine: Intracellular distribution, solubilization, purification and specificity

Abstract The intracellular distribution of disaccharidase activity was compared with that of acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) in rabbit small intestine using classical tissue-fractionation procedures and electron microscopy in conjunction with histochemical techniques. The disaccharidase activity was found to be concentrated in the “nuclear” fraction; acid phosphatase activity was distributed differently. In view of the methodological difficulties, definite conclusions on the true intracellular locus of disaccharidase activity were not drawn. Disaccharidase solubilization was achieved by sonication and subsequent extraction with n-butanol. Studies of enzyme specificity were conducted with an 18-fold purified enzyme preparation. Maltose was found to competitively inhibit sucrose cleavage suggesting a common enzyme-binding site.

Metabolism of connective tissue polysaccharides in vivo: IV. The sulphate group

Abstract The incorporation of inorganic sulphate into the chodroitin sulphate and keratosulphate components of rabbit costal cartilage and nucleus pulpsus has been studied. Both the soluble protein-polysaccharide complex as well as the insoluble residue exhibited rapid uptake of sulphate with significant incorporation occuring as early as 4 h after administration of the isotope. A procedure was advised for resolution of the keratosulphate and chondroitin sulphate fractions utilizing hyaluronidase digestion followed by chromatographys on DEAE-Sephadex in the presence of cetyltrimethylammonium bromide. The specific activity of the sulphate group in these polysaccharides was substantially unchanged for at least 30 days suggesting that the major portion of the sulphated polysaccharides of these tissues have relatively long biological half-lives. A high specific activity peak observed during several fractionation procedures was ultimately identified as inorganic sulphate. It is of interest to note that this material contaminated the polysaccharide fraction throughout several fractionation schemes. It is suggested that binding inorganic sulphate in tissue such as cartilage may occur via chelation with divalent cations which are tightly held by the ester sulphate groups on the polysaccharide moieties.