Bizzozero O A, McGarry J F, Lees M B
Biochemistry Department, E. K. Shriver Center, Waltham, Massachusetts 02254.
J Biol Chem. 1987 Oct 5;262(28):13550-7.
Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.
已知大鼠脑髓鞘蛋白脂蛋白（PLP）含有长链的、共价结合的脂肪酸。在表征酰化机制的过程中，我们发现分离得到的PLP在没有任何膜组分的情况下，与[3H]棕榈酰辅酶A（CoA）孵育后会发生酯化反应。这一观察结果表明该蛋白质既作为酰化酶又作为受体。因此，酰化是通过自催化过程发生的。通过向反应混合物中添加脑亚细胞组分以及改变分离程序，基本上排除了与PLP共纯化的单独酰基转移酶的可能性。脱酰化后，该蛋白质的酰化速率提高了4倍，这表明原来的位点被重新酰化。棕榈酰辅酶A浓度遵循米氏动力学，证实没有发生自发酰化。脉冲追踪实验表明该反应需要净添加酰基。尽管脂肪酸通过O - 酯键结合，但反应中需要游离的SH基团。用十二烷基硫酸钠或加热使蛋白质变性会抑制反应，而浅蓝菌素几乎没有影响。外周神经髓鞘中的主要蛋白质PO也是一种酰化蛋白质，但用[3H]棕榈酰辅酶A孵育外周髓鞘或分离出的蛋白质时它没有被标记，这表明它是通过不同途径进行酰化的。从PLP序列衍生的几种在体内已知被酰化位点的合成肽以及一系列脱酰化的PLP胰蛋白酶肽都没有被标记，这表明酰化需要蛋白质的完整性。有限的蛋白酶解和肽图谱分析表明，体外或体内的酰化位点相同，这表明自催化酰化反应是生理性的。
