Ostman A, Andersson M, Bäckström G, Heldin C H
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
J Biol Chem. 1993 Jun 25;268(18):13372-7.
Platelet-derived growth factor (PDGF)-BB is a dimeric protein held together by two disulfide bonds involving the 2nd and 4th cysteine residues from the NH2 terminus. To localize the three intrachain disulfide bonds in PDGF, a method was devised that made it possible to cleave PDGF at specific sites. A set of PDGF derivatives in which specific amino acids were mutated to methionine residues was generated. The recombinant proteins, immunoprecipitated from metabolically labeled transfected COS cells, were then subjected to CNBr cleavage and analyzed by SDS-gel electrophoresis under nonreducing conditions. Based on whether the mutated proteins remained in one piece or fell apart after CNBr cleavage, it was possible to deduce the disulfide bond arrangement in the PDGF B-chain; one bond involves the 1st and 6th cysteine residues, another the 3rd and 7th, and the last the 5th and 8th. The latter disulfide bond was found to be dispensable for receptor binding, whereas the former two were found to be essential for the correct folding or stability of the PDGF B-chain.
血小板衍生生长因子（PDGF）-BB是一种由两个二硫键连接在一起的二聚体蛋白，这两个二硫键涉及氨基末端的第2个和第4个半胱氨酸残基。为了定位PDGF中的三个链内二硫键，设计了一种方法，该方法能够在特定位点切割PDGF。生成了一组特定氨基酸突变为甲硫氨酸残基的PDGF衍生物。从代谢标记的转染COS细胞中免疫沉淀出重组蛋白，然后进行溴化氰切割，并在非还原条件下通过SDS凝胶电泳进行分析。根据突变蛋白在溴化氰切割后是保持完整还是分解，可以推断出PDGF B链中的二硫键排列；一个键涉及第1个和第6个半胱氨酸残基，另一个涉及第3个和第7个，最后一个涉及第5个和第8个。发现后一个二硫键对于受体结合是可有可无的，而前两个二硫键对于PDGF B链的正确折叠或稳定性是必不可少的。
