Guajardo Margarita H, Terrasa Ana M, Catalá Angel
Cátedra de Bioquímica, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina.
J Pineal Res. 2006 Oct;41(3):201-10. doi: 10.1111/j.1600-079X.2006.00352.x.
The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe2+ induced), during different time intervals (0-180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 +/- 2.9% in the native membranes to 12.65 +/- 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 +/- 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe2+ -ascorbate, we observed a decrease of protein thiols from 50.9 +/- 3.38 in native membranes to 1.72 +/- 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 +/- 2.50 to 12.50 +/- 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation.
视杆细胞外段（ROSg）膜本质上是脂蛋白复合物。视紫红质是ROSg的主要整合蛋白，被高度富含二十二碳六烯酸（22:6 n3）的磷脂所包围。这种流体环境对光激活后的构象变化起着重要作用。因此，ROSg膜极易受到氧化损伤。松果体、视网膜和其他组织中合成的褪黑素是一种自由基清除剂。这项工作的主要目的是研究从牛视网膜分离的ROSg膜在不同时间间隔（0 - 180分钟）内，经受非酶促脂质过氧化（抗坏血酸 - Fe2+诱导）后的变化。通过化学发光增加和脂肪酸改变来监测氧化应激。此外，我们研究了5 mM褪黑素的体外保护作用。发现在褪黑素存在下孵育的那些膜中，源自发光（化学发光）的总计数每分钟衰变数较低。当膜受到氧化损伤时，二十二碳六烯酸含量显著下降。这种下降从天然膜中的35.5±2.9%降至在180分钟内过氧化的膜中的12.65±1.86%。在5 mM褪黑素存在下，我们在过氧化的同时观察到22:6 n3的含量保持在23.85±2.77%。同时，使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）研究了氧化应激下膜蛋白的变化。蛋白质巯基的损失和羰基掺入的增加被用作蛋白质氧化的生物标志物。在暴露于Fe2+ - 抗坏血酸的膜中，我们观察到脂质过氧化180分钟后，蛋白质硫醇从天然膜中的50.9±3.38降至1.72±2.81 nmol/mg蛋白质，同时蛋白质中羰基的掺入从7.20±2.50增加到12.50±1.12 nmol/mg蛋白质。在SDS - PAGE中，我们观察到由于过氧化，所有蛋白质的含量下降，主要是视紫红质。褪黑素可防止脂质过氧化和蛋白质氧化。
