Yang J C, Chang P C, Fujitaki J M, Chiu K C, Smith R A
Biochemistry. 1986 May 6;25(9):2677-81. doi: 10.1021/bi00357a058.
Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.
所提供的证据表明磷脂与牛髓鞘碱性蛋白存在共价连接。对从经32P磷酸化的全髓鞘中获得的髓鞘碱性蛋白进行了磷脂部分的部分表征，该全髓鞘首先通过两次乙醚/乙醇（3:2 v/v）萃取、乙醚萃取和丙酮萃取进行脱脂，然后通过制备型十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳进行纯化。髓鞘碱性蛋白用丙酮水溶液沉淀并用蛋白酶处理。用羧肽酶Y或胰蛋白酶处理数小时会释放出一个亲脂性片段，该片段通过反相高效液相色谱纯化得到两种“脂肽”。这种脂肽可从大鼠和牛脑的主要和次要髓鞘碱性蛋白中获得。用弱碱或磷脂酶C处理可去除肽片段的亲脂性特征。该亲脂性片段是磷脂酶D的底物，但在薄层色谱上它与从用[γ-32P]ATP孵育的髓鞘中获得的任何32P标记的脂质不共迁移。已证明用弱酸处理经有机溶剂萃取并随后通过SDS-聚丙烯酰胺凝胶电泳纯化的髓鞘碱性蛋白会释放多磷酸肌醇。连同在脂肽的部分酸水解产物中鉴定出肌醇单磷酸这一事实，我们得出结论，多磷酸肌醇（磷脂酰肌醇4-磷酸和/或磷脂酰肌醇4,5-二磷酸）是脂肽的原始磷脂部分。
