Bronckers A L, van Elk R, Lyaruu D M, Wöltgens J H
J Biol Buccale. 1984 Sep;12(3):211-23.
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.
对体外完整的仓鼠磨牙牙胚进行了三步提取程序，这些牙胚用(32PO4)和/或(3H - 脯氨酸标记，以便分别量化牙本质基质（胶原蛋白）和富含脯氨酸的釉质基质蛋白的合成。该提取基于富含脯氨酸的釉质基质蛋白与相对不溶性的牙本质基质胶原蛋白相比具有高溶解性。用(10%)三氯乙酸预处理（步骤1）使牙胚脱矿并去除未掺入的氨基酸和/或小尺寸肽。连续水提取（步骤2）去除了通过SDS - 尿素 - 聚丙烯酰胺 - 电泳和氨基酸分析评估的大部分磷酸化釉原蛋白。胶原酶消化率数据表明该提取物中仅存在少量胶原蛋白。用(10%)甲酸进一步提取（步骤3）仅从外植体中释放少量釉原蛋白，但也增加了胶原蛋白和另一种主要是低分子成分的污染。残留在残渣中的大部分(3H)活性存在于胶原酶不稳定的物质中，被认为是牙本质胶原蛋白产生的合适测量方法。另一方面，残渣中还含有少量具有与釉原蛋白相同电泳迁移率的(3H)标记物质，但比水和甲酸提取物中的釉原蛋白具有更多的(32P)活性。有人认为残渣中的这种物质可能含有晶体结合的釉质基质蛋白。
