Divald Andras, Powell Saul R
Department of Medicine, Institute for Medical Research, North Shore-Long Island Jewish Health System, New Hyde Park, NY 11042, USA.
Free Radic Biol Med. 2006 Jan 1;40(1):156-64. doi: 10.1016/j.freeradbiomed.2005.09.022. Epub 2005 Oct 17.
Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.
已知心肌缺血/再灌注后会有多种蛋白质丢失，但对于介导的蛋白酶却知之甚少。本研究检验了蛋白酶体在清除心肌缺血期间氧化的蛋白质中起重要作用这一假说。通过用含2 μmol/L乳胞素的缓冲液灌注离体大鼠心脏10分钟来抑制蛋白酶体，这导致20S和26S蛋白酶体活性分别降低51%和42%，且至少持续90分钟。乳胞素预处理对经历30分钟全心缺血和60分钟再灌注的离体心脏的缺血后恢复影响较小。乳胞素预处理的缺血心脏的蛋白质羰基含量显著增加（P < 0.05）。已知一条分子量约为50 kDa的条带含有氧化肌动蛋白。通过分析3-甲基组氨酸对肌动蛋白降解进行定量，在30分钟缺血和60分钟再灌注后，3-甲基组氨酸显著减少（P < 0.05）15%。用乳胞素预处理缺血心脏可防止大部分3-甲基组氨酸的丢失（-6.5%）。用针对泛素的特异性抗体探测免疫沉淀的肌动蛋白，未发现含有该蛋白质泛素化同源物的条带。这些观察结果支持以下结论：蛋白酶体介导心肌缺血/再灌注期间氧化的一些蛋白质的清除，并且至少氧化肌动蛋白是由20S蛋白酶体清除的。
