Ueno A, Hong Y M, Arakaki N, Takeda Y
J Biochem. 1985 Aug;98(2):269-78. doi: 10.1093/oxfordjournals.jbchem.a135280.
A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496.
建立了一种从牛血清白蛋白胰蛋白酶消化物中半制备规模分离具有胰岛素刺激活性的低分子量多肽的方法，且该多肽具有明显的均一性。通过脂肪组织外植体测定法监测这种胰岛素刺激肽（ISP）的纯化过程，该测定法用于测量胰岛素对葡萄糖合成脂肪酸的刺激作用。通过DEAE - 纤维素柱色谱、Bio - Gel P - 10凝胶过滤、半制备C18反相HPLC柱上的疏水色谱以及SP - 5PW HPLC柱上的离子交换色谱相结合的方法对该多肽进行纯化。推导了ISP的一级结构。ISP是一种由71个氨基酸残基组成的双链多肽，基本上对应于牛血清白蛋白的115 - 143位和144 - 184（185）位残基通过二硫键相互连接。但是，将ISP的序列与Brown测定的牛血清白蛋白相关区域的序列进行比较发现，白蛋白的155和156位残基之间存在一个酪氨酸插入。因此，计算得出ISP的分子量为8496。
