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Identification of new rat dentin proteoglycans utilizing C18 chromatography.

Although only one small PG has been identified in dentin until now, a preliminary investigation has shown indications of the presence of several new proteoglycans (PGs) in rat incisor dentin. The aim of the present investigation was to isolate and characterize these PGs, which were labeled with 35S to facilitate the analysis. C18 chromatography resolved five dentin PGs. Based on SDS-polyacrylamide gel electrophoresis, their size varied from 100 to 400 kDa. The core proteins of the first four PGs appeared as 25, 40, 70, and 115 kDa bands. They stained turquoise with Stains All but did not stain with Coomassie Brilliant Blue. The core protein of the fifth PG appeared at about 45 kDa. This core protein stained with Coomassie Brilliant Blue but not with Stains All. In all PGs, the glycosaminoglycans consisted mainly of chondroitin 4-sulfate. To investigate their incorporation into predentin (young dentin that is not yet mineralized) and dentin, rat dentin PGs were pulse-labeled by injecting rats with [35S]sulfate at 5, 28, 55, or 177 h before killing the animals. Radiolabeling of predentin PGs was highest after 5 h and decreased rapidly (76%) over the next 50 h. In dentin PGs, a large percentage (34%) of the final quantity of incorporated 35S (at 177 h) was already present at 5 h. C18 chromatography of dentin PGs for each of the four time intervals showed similar 35S distribution patterns representing all five PGs, whereas the predentin appeared to contain mainly the fifth PG. This study demonstrates the existence of several apparently novel PGs in dentin that can be resolved by the use of a new method. These PGs were found in mineralized dentin and are thought to be rapidly transported toward the mineralization front. Part of the predentin PGs, on the other hand, seems to be lost as mineralization proceeds.