MacLean Isabelle A, Varma Anchal, Storey Kenneth B
Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Mol Cell Biochem. 2023 Feb;478(2):415-426. doi: 10.1007/s11010-022-04516-y. Epub 2022 Jul 8.
NADP-dependent isocitrate dehydrogenase (NADP-IDH, EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with the concomitant production of NADPH. NADPH plays important roles in many biosynthesis pathways, maintenance of proper oxidation-reduction balance, and protection against oxidative damage. This present study investigated the dynamic nature of NADP-IDH during hibernation by purifying it from the skeletal muscle of Richardson's ground squirrel (Urocitellus richardsonii) and analyzing its structural and functional changes in response to hibernation. Kinetic parameters of purified NADP-IDH from euthermic and hibernating ground squirrel skeletal muscle were characterized at 22 °C and 5 °C. Relative to euthermic muscle, -NADP-IDH in hibernating muscle had a higher affinity for its substrate, isocitrate at 22 °C, whereas at 5 °C, there was a significant decrease in isocitrate affinity. Western blot analysis revealed greater serine and threonine phosphorylation in hibernator NADP-IDH as compared to euthermic NADP-IDH. In addition, Bioinformatic analysis predicted the presence of 18 threonine and 21 serine phosphorylation sites on squirrel NADP-IDH. The structural and functional changes in NADP-IDH indicate the ability of the organism to reduce energy consumption during hibernation, while emphasizing increased NADPH production, and thus antioxidant activity, during torpor arousal cycles.
烟酰胺腺嘌呤二核苷酸磷酸（NADP）依赖的异柠檬酸脱氢酶（NADP-IDH，EC 1.1.1.42）催化异柠檬酸氧化脱羧生成α-酮戊二酸，同时产生NADPH。NADPH在许多生物合成途径、维持适当的氧化还原平衡以及抵御氧化损伤中发挥着重要作用。本研究通过从理查森地松鼠（Urocitellus richardsonii）的骨骼肌中纯化NADP-IDH并分析其在冬眠过程中的结构和功能变化，研究了冬眠期间NADP-IDH的动态特性。在22℃和5℃下对来自正常体温和冬眠地松鼠骨骼肌的纯化NADP-IDH的动力学参数进行了表征。相对于正常体温的肌肉，冬眠肌肉中的NADP-IDH在22℃时对其底物异柠檬酸具有更高的亲和力，而在5℃时，异柠檬酸亲和力显著降低。蛋白质免疫印迹分析显示，与正常体温的NADP-IDH相比，冬眠动物的NADP-IDH中丝氨酸和苏氨酸磷酸化程度更高。此外，生物信息学分析预测松鼠NADP-IDH上存在18个苏氨酸和21个丝氨酸磷酸化位点。NADP-IDH的结构和功能变化表明，生物体在冬眠期间有能力降低能量消耗，同时强调在蛰伏觉醒周期中增加NADPH的产生，从而增强抗氧化活性。
