Detection of mature collagen in human dental enamel.

Mature dental enamel is the most mineralized of all mammalian tissues and considered to be free of collagen. Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are two nonreducible cross-links of mature collagen. Hydroxyproline (Hyp) is an amino acid that is believed to be indicative of the presence of collagen. We set out to assess the concentrations of Hyp, HP, and LP in dental enamel and dentin (control) to clarify whether there was minor collagen content in dental enamel. We studied 17.53 g of enamel and 22.12 g of dentin gained from 120 extracted human teeth. Enamel and dentin (control) were separated with a diamond dental drill under microscopic control by wasting a margin of enamel (Ca. 2 mm) at the dentin-enamel border. Collagen alpha-chains were analyzed by Sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) after decalcification and collagen extraction. Concentrations of HP and LP where measured by using high-performance liquid chromatography (HPLC). Hyp was analyzed by a spectrophotometric method. The pooled probe of enamel contained 0.23 mug/g of Hyp. This concentration was 49 times lower than that in dentin. Concentrations of HP and LP in enamel were 0.07 nmol/g and 0.02 nmol/g, respectively being 605.57 (HP) and 251.50 (LP) times lower in enamel as compared to dentin. Collagen type I was found in enamel; collagen types I and V were found in dentin samples. In reports of many studies and textbooks, collagen is considered to be completely absorbed in the course of the mineralization and maturation of dental enamel. We show that this is not the case. However, the concentration of collagen in enamel was considerably lower as compared to that in dentin.

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1).

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.