Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts.
Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts.
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牙本质的非胶原蛋白。
DOI：
10.1016/s0021-9258(19)70720-0
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
W. Butler
中科院分区：
文献类型：
--
作者：
A. Linde;M. Bhown;W. Butler
使用多种预防措施从大鼠牙本质中获得非胶原蛋白（NCP），以防止蛋白质的人为降解和损失。在脱矿质之前，用含有酶抑制剂的 4 M 盐酸胍 (GdmCl) 提取大鼠切牙牙本质。用 GdmCl 提取的唯一主要成分是蛋白聚糖组分。当用含有蛋白酶抑制剂的 EDTA 溶液对门牙进行脱钙时，大部分 NCP 被提取出来。 EDTA提取物含有四种类型的大分子：酸性糖蛋白、含γ-羧基谷氨酸(Gla)的蛋白质、磷蛋白和蛋白聚糖。除两个例外外，这些 NCP 的表观分子量均大于 50,000。我们的观察结果与其他人获得的人类牙本质 NCP 的结果形成鲜明对比，表明在这些先前的研究中发生了一些 NCP 的人为降解和损失。当 EDTA 提取物在 DEAE-纤维素上进行色谱分离时，含有机磷酸盐的级分呈双相。通过两种不同的程序对该级分进行重色谱分离，将材料分离成含有复杂糖蛋白的级分和单个大鼠门牙磷蛋白峰（RIP）。因此，其他人早期关于含 RIP 级分的双相性质代表两种截然不同的磷蛋白种类的解释还为时过早。通过磺化聚苯乙烯柱制备的高度纯化的 RIP 不含半胱氨酸、缬氨酸、蛋氨酸、亮氨酸、苯丙氨酸或精氨酸，并且磷酸丝氨酸残基的水平高于以前的任何报告。当磷蛋白制剂被去磷酸化（dP-RIP）并在 DEAE-纤维素上重新层析时，观察到部分分离成两个级分。 dP-RIP 级分的自动 Edman 降解表明存在两个 NH2 末端序列：Asp-Asp-Asp-Asn 和 Asp-Asp-Pro-Asn。然而，当将该材料应用于7.5%十二烷基硫酸钠聚丙烯酰胺凝胶电泳并用考马斯亮蓝染色时，显示出单一蛋白质条带。与标准球状蛋白相比，dP-RIP 的表观分子量约为 72,000。数据表明，大鼠牙本质至少含有两种主要的 RIP 分子种类，它们的大小和结构密切相关。此外，初步证据表明可能存在其他次要形式的相关磷蛋白。
Noncollagenous proteins (NCPs) were obtained rrom rat dentin using several precautionary measures to prevent artifactual degradation and losses of the proteins. Prior to demineralization, rat incisor dentin was extracted with 4 M guanidine hydrochloride (GdmCl) containing enzyme inhibitors. The only major component extracted with GdmCl was a proteoglycan fraction. Most of the NCPs were extracted when the incisors were decalcified with an EDTA solution containing protease inhibitors. The EDTA extract contained four types of macromolecules: acidic glycoproteins, gamma-carboxyglutamic acid (Gla)-containing proteins, phosphoproteins, and proteoglycans. With two exceptions, the apparent molecular weights of these NCPs were greater than 50,000. Our observations contrast sharply with the results obtained by others for human dentin NCPs and suggest that artifactual degradation and losses of some NCPs occurred in these previous studies. The organic phosphate-containing fraction was biphasic when the EDTA extract was chromatographed on DEAE-cellulose. Rechromatography of this fraction by two different procedures separated the material into a complex glycoprotein-containing fraction and, a single rat incisor phosphoprotein peak (RIP). Thus, the earlier interpretation by others that the biphasic nature of the RIP-containing fraction represents two widely differing phosphoprotein species was premature. Highly purified RIP, prepared by passage through a sulfonated polystyrene column, contained no cysteine, valine, methionine, leucine, phenylalanine, or arginine, and the level of phosphoseryl residues was higher than for any previous report. When this preparation of phosphoprotein was dephosphorylated (dP-RIP) and rechromatographed on DEAE-cellulose, a partial separation into two fractions was observed. Automated Edman degradation of fraction dP-RIP suggested the presence of two NH2-terminal sequences: Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. However, this material displayed a single protein band when applied to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The apparent molecular weight of dP-RIP, compared to standard globular proteins, was about 72,000. The data suggest that rat dentin contains at least two major molecular species of RIP that are closely related in size and-structure. In addition, preliminary evidence suggested that other minor forms of related phosphoproteins may exist.