Sipione S, Lupo G, Anfuso C D, Albanese V, Alberghina M
Institute of Biochemistry, Faculty of Medicine, University of Catania, Italy.
FEBS Lett. 1996 Apr 8;384(1):19-24. doi: 10.1016/0014-5793(96)00270-0.
In microvessels isolated from bovine brain, microsomal enzyme activities involved in phosphatidylcholine biosynthesis and degradation were determined. The microvessels possessed acyl-CoA:1-acyl-sn-glycero-3-phosphocholine (AT) and glycerophosphocholine phosphodiesterase (GroPChoPDE) activity at a higher level compared with bovine and rat brain or rat liver microsomes whereas they expressed CTP:phosphocholine cytidylyltransferase (CT) and choline phosphotransferase (CPT) activity at a lower level. Each enzyme has been characterized in terms of response to inhibitors or activators revealing properties very similar to those in brain and liver microsomes. In the homogenate prepared from t-butylhydroperoxide-treated microvessels (10 min exposure to 10 microM up to 1 mM concentrations), AT and CPT activities exhibited a significant dose-dependent inhibition. In contrast, GroPChoPDE activity was unaffected. CT was inhibited only at 1 mM concentration. Short treatment of microvessels with Fe2+ (20 microM)-ascorbate (0.25 mM) or 100 microM linoleate hydroperoxide did not have any effect on the activity of the four enzymes. Strong inhibition of all enzymes was noted when the linoleate hydroperoxide system was fortified by Fe2+ ions (100 microM). AT inactivation was also found when oxidized low density lipoprotein was preincubated with microvessels. On the other hand, oxidized LDL left unchanged CPT and GroPChoPDE activities whereas it promoted a slight stimulation of cytidylyltransferase activity. Overall, the results suggest a link between oxygen radical generation and the perturbation of the microvessel membrane structure in which the four enzymes are incorporated, coupled to a direct sulfhydryl protein modification.
在从牛脑中分离出的微血管中，测定了参与磷脂酰胆碱生物合成和降解的微粒体酶活性。与牛脑、大鼠脑或大鼠肝脏微粒体相比，微血管中酰基辅酶A：1-酰基-sn-甘油-3-磷酸胆碱（AT）和甘油磷酸胆碱磷酸二酯酶（GroPChoPDE）的活性较高，而其CTP：磷酸胆碱胞苷转移酶（CT）和胆碱磷酸转移酶（CPT）的活性较低。每种酶已根据对抑制剂或激活剂的反应进行了表征，其特性与脑和肝脏微粒体中的非常相似。在由叔丁基过氧化氢处理的微血管制备的匀浆中（暴露于10微摩尔至1毫摩尔浓度下10分钟），AT和CPT活性表现出显著的剂量依赖性抑制。相比之下，GroPChoPDE活性不受影响。CT仅在1毫摩尔浓度下受到抑制。用Fe2+（20微摩尔）-抗坏血酸盐（0.25毫摩尔）或100微摩尔亚油酸氢过氧化物对微血管进行短时间处理对这四种酶的活性没有任何影响。当亚油酸氢过氧化物体系用Fe2+离子（100微摩尔）强化时，所有酶都受到强烈抑制。当氧化低密度脂蛋白与微血管预孵育时，也发现了AT失活。另一方面，氧化低密度脂蛋白使CPT和GroPChoPDE活性保持不变，而它促进了胞苷转移酶活性的轻微刺激。总体而言，结果表明氧自由基生成与包含这四种酶的微血管膜结构扰动之间存在联系，这与直接的巯基蛋白修饰有关。
