Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone.
Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone.
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DOI：
10.1016/s0021-9258(18)61592-3
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发表时间：
1987-02
期刊：
影响因子：
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通讯作者：
C. Prince;T. Oosawa;W. Butler;M. Tomana;A. Bhown;M. Bhown;R. Schrohenloher
中科院分区：
文献类型：
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作者：
C. Prince;T. Oosawa;W. Butler;M. Tomana;A. Bhown;M. Bhown;R. Schrohenloher
磷酸化糖蛋白是从用0.5 M EDTA溶于4 M盐酸胍中的大鼠骨脱矿提取的蛋白质混合物中纯化的。一个高水平的纯度的制备表明由一个单一的带上的十二烷基硫酸钠（SDS）-梯度凝胶电泳，沉降平衡ultracentralgal数据，并通过自动Edman降解结果。通过在4 M氯化胍中的沉降平衡分析，磷蛋白的分子量显示为约44，000，尽管通过5- 15%SDS-聚丙烯酰胺凝胶电泳获得的Mr为75，000。随后通过15%SDS-聚丙烯酰胺凝胶电泳分析得到45，000的Mr。分析数据表明，该蛋白含有16.6%的碳水化合物，可能包括1个N-连接的寡糖和5-6个O-连接的寡糖。富含天冬氨酸和谷氨酸的蛋白质含有约300个氨基酸残基，包括1个磷酸苏氨酸和12个磷酸丝氨酸残基。碱性β-消除/NaBH 4还原数据表明，在氨基酸分析之前通过完全酸水解获得的磷酸盐与碱性β-消除的磷酸盐相当。在该实验中，丝氨酸加苏氨酸的损失超过磷酸盐释放量5-6个残基/蛋白质。这些丝氨酸和苏氨酸残基可能代表O-连接的寡糖，因为蛋白质含有大约这个数量的N-乙酰基-半乳糖胺残基。克隆性大鼠骨肉瘤细胞培养显示，成骨样细胞合成和分泌磷蛋白。用32 PO 4脉冲后，分泌到培养基中的蛋白质用三氯乙酸沉淀，放射性标记的蛋白质被免疫吸附。在5- 15%SDS-聚丙烯酰胺凝胶电泳（即Mr = 75，000）和15%凝胶（Mr = 45，000）上，与从骨中获得的磷蛋白在相同位置迁移的蛋白质可被特异性免疫沉淀。
A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.