Hermes-Lima M, Storey K B
Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
Z Naturforsch C J Biosci. 1995 Sep-Oct;50(9-10):685-94. doi: 10.1515/znc-1995-9-1015.
During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods.
在陆地蜗牛乳白奥塔蜗牛从夏眠中苏醒的过程中，其活跃的代谢功能在数分钟内得以恢复，耗氧量急剧增加。在从夏眠的低氧状态转变为常氧状态的过程中，肝胰腺中的黄嘌呤氧化酶（XO）可能导致了所观察到的脂质过氧化现象。使用基于蝶呤氧化的荧光测定法，对肝胰腺提取物中XO以及XO + XDH（XO和黄嘌呤脱氢酶活性之和）的活性和一些特性进行了测定。XO和XO + XDH对蝶呤的米氏常数（Km）分别为9微摩尔和6微摩尔，而XDH对亚甲蓝的Km为5微摩尔。XO + XDH和XO的活性均受到别嘌呤醇（半数抑制浓度I50 = 2微摩尔）、40℃预孵育以及5分钟过氧化氢预暴露的抑制。在反应中加入叠氮化物，由于抑制了高内源性过氧化氢酶活性，使得过氧化氢的失活能力提高了约70倍。在有叠氮化物存在的情况下，XO + XDH和XO活性对过氧化氢的I50分别为0.04毫摩尔和0.11毫摩尔。与哺乳动物的XO情况不同，乳白奥塔蜗牛的XO（由蝶呤）预先还原对于使该酶易受过氧化氢影响并非必要。有趣的是，亚甲蓝部分地防止了热和过氧化氢诱导的XO + XDH活性失活。这些数据表明，酶 - 亚甲蓝复合物的形成诱导了对黄素腺嘌呤二核苷酸（FAD）活性位点的热和氧化损伤的保护作用。与从休眠中苏醒24小时后的活跃蜗牛相比，夏眠35天后的蜗牛中XO和XO + XDH的活性均显著更高。在夏眠的乳白奥塔蜗牛中，XO /（XO + XDH）活性的比值也略有增加（从0.07增至0.09；P < 0.025）。在夏眠蜗牛中，XO活性为0.03纳摩尔·分钟⁻¹·毫克蛋白⁻¹。与肝胰腺过氧化氢酶相比，XO活性可能过低，无法对氧自由基的净生成以及因此对过氧化损伤做出显著贡献。相反，XO诱导氧化应激的低可能性可能构成乳白奥塔蜗牛在苏醒期的一种适应性优势。
