Institute of Biochemistry, Department of Biology and Department of Chemistry , Carleton University , Canada.
PeerJ. 2013 Feb 12;1:e12. doi: 10.7717/peerj.12. Print 2013.
Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, plays a crucial role both in sustaining glycolytic ATP production under oxygen-limiting conditions and in facilitating the catabolism of accumulated lactate when stress conditions are relieved. In this study, the effects on LDH of in vivo freezing and dehydration stresses (both of which impose hypoxia/anoxia stress on tissues) were examined in skeletal muscle of the freeze-tolerant wood frog, Rana sylvatica. LDH from muscle of control, frozen and dehydrated wood frogs was purified to homogeneity in a two-step process. The kinetic properties and stability of purified LDH were analyzed, revealing no significant differences in V max, K m and I 50 values between control and frozen LDH. However, control and dehydrated LDH differed significantly in K m values for pyruvate, lactate, and NAD, I 50 urea, and in temperature, glucose, and urea effects on these parameters. The possibility that posttranslational modification of LDH was responsible for the stable differences in enzyme behavior between control and dehydrated states was assessed using ProQ diamond staining to detect phosphorylation and immunoblotting to detect acetylation, methylation, ubiquitination, SUMOylation and nitrosylation of the enzyme. LDH from muscle of dehydrated wood frogs showed significantly lower levels of acetylation, providing one of the first demonstrations of a potential role for protein acetylation in the stress-responsive control of a metabolic enzyme.
乳酸脱氢酶（LDH）是无氧糖酵解的终末酶，在缺氧/缺氧应激条件下，它在维持糖酵解 ATP 产生和缓解应激条件时促进积累的乳酸分解代谢方面发挥着关键作用。在这项研究中，研究了在耐冻木蛙（Rana sylvatica）的骨骼肌中，体内冷冻和脱水应激（这两种应激都会对组织造成缺氧/缺氧应激）对 LDH 的影响。采用两步法从对照、冷冻和脱水木蛙的肌肉中纯化 LDH，达到均一性。分析了纯化 LDH 的动力学特性和稳定性，发现对照和冷冻 LDH 的 V max、K m和 I 50值没有显著差异。然而，对照和脱水 LDH 的丙酮酸、乳酸和 NAD、I 50 尿素的 K m值以及温度、葡萄糖和尿素对这些参数的影响存在显著差异。使用 ProQ diamond 染色检测磷酸化，免疫印迹检测乙酰化、甲基化、泛素化、SUMO 化和亚硝基化，评估 LDH 翻译后修饰是否是导致对照和脱水状态下酶行为稳定差异的原因。脱水木蛙肌肉中的 LDH 显示出明显较低水平的乙酰化，这是首次证明蛋白质乙酰化在代谢酶的应激响应控制中可能发挥作用。
