Bizzozero O A, Lees M B
J Neurochem. 1986 Feb;46(2):630-6. doi: 10.1111/j.1471-4159.1986.tb13013.x.
The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.
在体外系统中确定了蛋白脂蛋白（PLP）脂肪酸酯化反应的直接酰基链供体。去除粗核和线粒体部分后的大鼠脑总膜，与放射性酰基供体一起孵育，用氯仿/甲醇提取，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析。在[3H]棕榈酸、辅酶A、ATP和Mg2+存在的情况下，内源性PLP的酰化以线性速率进行至少2小时。放射性通过酯键与蛋白质结合，主要以棕榈酸形式存在。省略ATP、辅酶A、Mg2+或全部三种物质会使脂肪酸掺入PLP的量分别降至完整系统中值的44%、27%、8%和4%。在没有辅酶A和ATP的情况下，用[3H]棕榈酰辅酶A孵育膜部分会导致PLP高度标记。这些数据表明酰化需要游离脂肪酸的激活。磷脂和糖脂不能直接使PLP酰化。最后，当在没有辅因子的情况下将分离的髓磷脂与[3H]棕榈酰辅酶A一起孵育时，只有PLP被标记，从而证实了棕榈酰辅酶A作为直接酰基链供体的身份，并表明酰化活性和可用于酰化的PLP池都在髓磷脂中。
