Zhang Z, Blake D R, Stevens C R, Kanczler J M, Winyard P G, Symons M C, Benboubetra M, Harrison R
Bone and Joint Research Unit, St Bartholomew's and the Royal London School of Medicine and Dentistry, University of London, UK.
Free Radic Res. 1998 Feb;28(2):151-64. doi: 10.3109/10715769809065801.
Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.
黄嘌呤氧化酶（XO）传统上被认为是活性氧（ROS）的产生者，ROS会导致组织中的缺氧再灌注损伤。然而，由于人XO对黄嘌呤明显缺乏活性，且别嘌呤醇无法抑制再灌注损伤，因此其在人体内的这一作用存在争议。在本文中，我们采用天然凝胶电泳结合活性染色来研究人黄嘌呤脱氢酶（XD）和XO在缺氧再灌注损伤中的作用。这种方法提供了传统分光光度法无法获得的信息。我们发现，人脐静脉内皮细胞（HUVECs）和淋巴细胞白血病细胞（CEMs）中的XD和XO均通过氧化NADH而非次黄嘌呤来催化ROS的产生。在HUVECs和CEMs中均观察到了XD向XO的转化，尽管转化水平有所不同。纯化的人乳XD在存在NADH时比存在次黄嘌呤时更有效地产生ROS。这种NADH氧化活性被黄素腺嘌呤二核苷酸（FAD）位点抑制剂二苯基碘鎓（DPI）阻断，但不受钼位点抑制剂别嘌呤醇的抑制。然而，在同时存在DPI和别嘌呤醇的情况下，以NADH或次黄嘌呤为底物时，XD/XO的活性被完全阻断。我们得出结论，人XD和XO均可氧化NADH以产生活性氧。因此，缺血后ROS的产生并不需要XD向XO的转化。应重新评估缺氧再灌注损伤假说，以考虑XD和XO在氧化NADH以产生活性氧中所起的重要作用。
