The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole‐body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post‐translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a K_m value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta‐N‐acetylglucosamination, and cysteine nitrosylation, post‐translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration‐responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I₅₀ of 7.3 M, while the I₅₀ from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration‐induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.

中文翻译：

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注意 GAP：极端脱水过程中耐尿素 GAPDH 的纯化和表征

非洲爪蛙 ( Xenopus laevis ) 能承受长时间的全身极度脱水，导致血流受损、全身缺氧和缺血应激。在脱水期间，这些青蛙从氧化代谢转变为依赖无氧糖酵解。在这项研究中，我们将中心糖酵解酶甘油醛-3-磷酸脱氢酶 (GAPDH) 纯化为电泳均一性，并研究了对照和 30% 脱水的蟾蜍之间的结构、动力学、亚细胞定位和翻译后修饰特性肝脏。与来自水合青蛙的酶相比，来自脱水肝脏的 GAPDH 的最大速度降低了 25.4%，其对 GAP 的亲和力增加了 55.7%。在脱水模拟条件下（150 mM 尿素和 1% PEG），GAP 亲和力随K _m降低值比对照高 53.8%。青蛙脱水还诱导丝氨酸磷酸化、甲基化、乙酰化、β-N-乙酰氨基葡萄糖化和半胱氨酸亚硝基化、翻译后修饰 (PTM) 显着增加。这些修饰经过生物信息学预测和实验验证，以控制蛋白质稳定性、酶活性和核易位，这些在脱水过程中会增加。然而，这些脱水反应性蛋白质修饰似乎并没有影响酶的热稳定性，因为在用差示扫描荧光法测试时 GAPDH 熔解温度保持不变。PTM 可以促进脱水 GAPDH 中的极端尿素抗性，因为来自脱水动物的酶的尿素 I ₅₀为 7.3 M，而 I ₅₀来自水合酶的 5.3 M。 这些脱水诱导的 GAPDH 分子修饰的生理后果可能会抑制 GADPH 糖酵解功能在脱水X. laevis经历的循环减少和整体缺氧期间。