Gulati S, Ainol L, Orak J, Singh A K, Singh I
Department of Pediatrics, Medical University of South Carolina, Charleston 29425.
Biochim Biophys Acta. 1993 Oct 20;1182(3):291-8. doi: 10.1016/0925-4439(93)90071-8.
We have previously demonstrated that ischemic injury results in the loss of peroxisomal functions (e.g., inhibition of catalase activity and fatty-acid beta-oxidation activity). To understand the molecular mechanism leading to the loss of peroxisomal beta-oxidation in ischemic tissue, we examined the levels of individual enzyme activities and proteins of the peroxisomal beta-oxidation system and overall fatty-acid oxidation in peroxisomes isolated from kidney exposed to ischemia-reperfusion injury. The peroxisomal beta-oxidation decreased with an increase in time of ischemic injury (53% and 43% of the control in kidneys exposed to 60 and 90 min ischemia, respectively). In vivo inactivation of catalase with aminotriazole and exposure of isolated peroxisomes to H2O2 resulted in inhibition of peroxisomal beta-oxidation system suggesting that this enzyme system is labile to excessive H2O2 produced during ischemic injury. The enzyme activities of lignoceroyl-CoA ligase, acyl-CoA oxidase, bifunctional enzymes and acyl-CoA thiolase (individual peroxisomal beta-oxidation enzymes) after 90 min of ischemia were 87, 80, 87 and 85% of the control, respectively. This decrease in enzyme activities was more pronounced following reperfusion (28, 11, 23 and 35% of the control, respectively). Immunoblot analysis of these enzymes indicated that the major loss of these enzyme activities during ischemia was due to their inactivation, whereas during reperfusion, proteolysis also contributed toward the observed loss of these activities. In summary, these results demonstrated that loss of peroxisomal beta-oxidation in ischemia-reperfusion injury was due to inactivation and proteolysis of beta-oxidation enzymes. Acyl-CoA oxidase was more sensitive to ischemia-reperfusion injury compared to other enzymes, and the overall loss of peroxisomal beta-oxidation may be a reflection of the loss of acyl-CoA oxidase activity, a rate-limiting enzyme.
我们之前已经证明，缺血性损伤会导致过氧化物酶体功能丧失（例如，过氧化氢酶活性和脂肪酸β-氧化活性受到抑制）。为了了解缺血组织中过氧化物酶体β-氧化丧失的分子机制，我们检测了从遭受缺血再灌注损伤的肾脏中分离出的过氧化物酶体中过氧化物酶体β-氧化系统的各个酶活性和蛋白质水平以及整体脂肪酸氧化情况。随着缺血损伤时间的增加，过氧化物酶体β-氧化降低（分别暴露于60分钟和90分钟缺血的肾脏中，过氧化物酶体β-氧化分别为对照组的53%和43%）。用氨基三唑在体内使过氧化氢酶失活以及将分离出的过氧化物酶体暴露于过氧化氢会导致过氧化物酶体β-氧化系统受到抑制，这表明该酶系统对缺血损伤期间产生的过量过氧化氢不稳定。缺血90分钟后，木蜡酰辅酶A连接酶、酰基辅酶A氧化酶、双功能酶和酰基辅酶A硫解酶（过氧化物酶体β-氧化的各个酶）的酶活性分别为对照组的87%、80%、87%和85%。再灌注后，这些酶活性的降低更为明显（分别为对照组的28%、11%、23%和35%）。对这些酶的免疫印迹分析表明，缺血期间这些酶活性的主要丧失是由于它们的失活，而在再灌注期间，蛋白水解也导致了观察到的这些活性的丧失。总之，这些结果表明，缺血再灌注损伤中过氧化物酶体β-氧化的丧失是由于β-氧化酶的失活和蛋白水解。与其他酶相比，酰基辅酶A氧化酶对缺血再灌注损伤更敏感，过氧化物酶体β-氧化的整体丧失可能反映了限速酶酰基辅酶A氧化酶活性的丧失。
