Basavarajappa B S, Cooper T B, Hungund B L
Division of Analytical Psychopharmacology, New York State Psychiatric Institute, Orangeburg, NY 10962, USA.
Biochem Pharmacol. 1998 Feb 15;55(4):515-21. doi: 10.1016/s0006-2952(97)00501-7.
The enzyme phospholipase A2 (PLA2), which catalyzes the hydrolysis of an ester bond at the sn-2 position of 1,2-sn-diacylglycerols, has been suggested to play an important role in regulating cellular functions. Although ethanol (EtOH)-induced activation of PLA2 activity was reported previously by us in mouse brain (Hungund et al., Neurochem Int 25: 321-325, 1994), its subcellular localization and biochemical properties have not been investigated. Therefore, in the present study, we examined the subcellular localization and characterization of EtOH-activated PLA2 activity in mouse brain. The results indicated that EtOH treatment decreased the specific activity of PLA2 for the first 48 hr, and then the activity increased and reached a peak level in both cytosol (1.6-fold) and membrane (1.7-fold) fractions at 96 hr of exposure. Specific activity was found to be higher in the membrane fraction than in the cytosol. Using differential density gradient centrifugation, subcellular localization of the membrane-associated PLA2 revealed that most of the EtOH-activated PLA2 specific activity was associated with the synaptic membrane (44%) followed by the nuclear membrane (13%). No significant increase in the PLA2 specific activity of mitochondrial and microsomal membranes was observed. No activity was detected in the myelin membrane. PLA2 specific activity of membranes from control and EtOH-exposed mouse brain exhibited preference for arachidonic acid over linoleic acid at the sn-2 position of glycero-3-phosphocholine (PC). No detectable PLA2 specific activity was found when PC containing oleic acid at the sn-2 position was used as a substrate. The present results also indicated that the PLA2 specific activity of membrane from control and EtOH-exposed mouse brain was insensitive to dithiothreitol, strongly stimulated by Ca2+, enhanced by glycerol, and inhibited by the cytosolic PLA2 (cPLA2) inhibitor methyl arachidonyl fluorophosphonate with an IC50 value of 3.33 microM. In summary, results suggest that the properties of EtOH-activated PLA2 activity found in mouse brain membrane fraction are similar to those of cPLA2 found in variety of cells, including mammalian brain.
磷脂酶A2（PLA2）可催化1,2 - 二酰基甘油sn - 2位酯键的水解，有人认为它在调节细胞功能中起重要作用。尽管我们之前报道过乙醇（EtOH）可诱导小鼠脑内PLA2活性激活（洪贡德等人，《神经化学国际》25: 321 - 325, 1994），但其亚细胞定位和生化特性尚未得到研究。因此，在本研究中，我们检测了小鼠脑内乙醇激活的PLA2活性的亚细胞定位和特性。结果表明，乙醇处理在最初48小时降低了PLA2的比活性，然后活性增加，并在暴露96小时时在胞质溶胶（1.6倍）和膜（1.7倍）组分中达到峰值水平。发现膜组分中的比活性高于胞质溶胶。使用差速密度梯度离心法对膜相关PLA2进行亚细胞定位，结果显示大部分乙醇激活的PLA2比活性与突触膜相关（44%），其次是核膜（13%）。未观察到线粒体膜和微粒体膜的PLA2比活性有显著增加。在髓鞘膜中未检测到活性。对照和乙醇处理的小鼠脑膜的PLA2比活性在甘油 - 3 - 磷酸胆碱（PC）的sn - 位对花生四烯酸的偏好高于亚油酸。当使用在sn - 2位含有油酸的PC作为底物时，未检测到可检测的PLA2比活性。本研究结果还表明，对照和乙醇处理的小鼠脑膜的PLA2比活性对二硫苏糖醇不敏感，受Ca2 +强烈刺激，受甘油增强，并受胞质PLA2（cPLA2）抑制剂甲基花生四烯酰氟磷酸酯抑制，IC50值为3.33 microM。总之，结果表明在小鼠脑膜组分中发现的乙醇激活的PLA2活性特性与在包括哺乳动物脑在内的多种细胞中发现的cPLA2特性相似。
