Mueller Sebastian, Weber Angelika, Fritz Reiner, Mütze Sabine, Rost Daniel, Walczak Henning, Völkl Alfred, Stremmel Wolfgang
Department of Internal Medicine IV, University of Heidelberg, Bergheimer Strasse 58, 69115 Heidelberg, Germany.
Biochem J. 2002 May 1;363(Pt 3):483-91. doi: 10.1042/0264-6021:3630483.
Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H(2)O(2): an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H(2)O(2) turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H(2)O(2) generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H(2)O(2) can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H(2)O(2) forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H(2)O(2) in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the beta-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H(2)O(2) from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H(2)O(2) metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.
过氧化物酶体是必不可少且普遍存在的细胞器，在哺乳动物脂质和氧代谢中起关键作用。黄素氧化酶的存在使其成为细胞内重要的过氧化氢来源：这是过氧化物酶体氧化还原反应的必然产物，也是一种关键的活性氧。过氧化物酶体响应过氧化物酶体增殖激活受体信号通路触发的外部信号而增殖。这种增殖导致的过氧化物酶体衍生的氧化应激越来越被认为参与了从啮齿动物致癌到人类酒精性和非酒精性脂肪性肝炎等多种病理过程。迄今为止，尚无灵敏的方法实时记录过氧化物酶体的过氧化氢周转情况。在此，我们介绍一种灵敏的化学发光方法，可实时监测完整过氧化物酶体悬浮液中过氧化氢的生成和降解。重要的是，在同一样本中，可在纳摩尔无毒浓度下评估过氧化氢的去除以及释放情况。由于过氧化氢酶和氧化酶的动力学特性，在存在各种过氧化物酶体底物的情况下，过氧化氢会形成快速稳态浓度。底物筛选表明，尿酸盐、乙醇酸和活化脂肪酸是啮齿动物中过氧化氢的最重要来源。动力学研究进一步表明，过氧化物酶体除了在长链和极长链脂肪酸分解中起关键作用外，还对中链脂肪酸的β氧化有显著贡献。这些观察结果确定了完整过氧化物酶体中过氧化氢的直接定量释放。该实验方法为在真核细胞过氧化物酶体中功能研究过氧化氢代谢、底物转运和周转提供了新的可能性。
