Biosynthesis of tooth germ proteins in vitro: a fast quantitative extraction of amelogenins from intact hamster molar tooth germs.
A three step extraction procedure was carried out on intact hamster molar tooth germs in vitro labelled with 32PO4 and/or 3H-proline, in order to quantify separately the synthesis of dentine matrix (collagen) and the proline rich enamel matrix proteins. The extraction was based on the high solubility of the proline rich enamel matrix proteins compared with the relatively insoluble dentine matrix collagens. Pretreatment with 10% trichloroacetic acid (step 1) demineralized and removed the non-incorporated amino acids and/or small sized peptides. A consecutive water extraction (step 2) removed a large percentage of the phosphorylated amelogenins as assessed by SDS-urea-polyacrylamide-electrophoresis and amino acid analyses. Collagenase digestibility data showed that only small amounts of collagens were present in this extract. Further extraction with 10% formic acid (step 3) released only small amounts of amelogenins from the explants but also increased contamination with collagens and another predominantly low molecular components. Most of the 3H-activity remaining in the residues was found in the collagenase labile material and was considered to be an appropriate measure for production of dentine collagens. On the other hand, the residues also contained small amounts of 3H-labelled material with the same electrophoretic mobility as amelogenins but had much more 32P-activity than the amelogenins derived from the water and formic acid extracts. It is suggested that this material in the residues probably contains the crystal bound enamel matrix proteins.
D000566:Amelogenesis; D053523:Amelogenin; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006224:Cricetinae; D046508:Culture Techniques; D003746:Dental Enamel Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008647:Mesocricetus; D008722:Methods; D008963:Molar; D013997:Time Factors; D014083:Tooth Germ / Q000378:metabolism*
D053523:Amelogenin; D000596:Amino Acids; D003746:Dental Enamel Proteins