Mattice AMS et al. (2023), Role of NADP+-dependent isocitrate dehydrogenas...

XB-ART-59972

Biochimie 2023 Jul 01;210:14-21. doi: 10.1016/j.biochi.2023.03.008.

Show Gene links Show Anatomy links

Role of NADP+-dependent isocitrate dehydrogenase from muscle tissue of Rana sylvatica in ROS defense during freeze-tolerance.

Mattice AMS , Varma A , Storey KB .

???displayArticle.abstract???
The wood frog, Rana sylvatica, employs freeze tolerance as a winter survival strategy in seasonally cold environments. At subzero temperatures, up to 65-70% of total body water can freeze in extracellular spaces, halting vital functions (breathing, heartbeat) and causing ischemia that, in turn, can have numerous consequences including the generation of damaging reactive oxygen species (ROS). NADPH serves as a key donor of reductive power for most ROS detoxifying enzymes and can be generated by several metabolic pathways. One source of NADPH reducing power is the NADP-dependent isocitrate dehydrogenase (IDH) reaction. The present study evaluated the properties and regulation of IDH from skeletal muscle of R. sylvatica when frogs were exposed to stress conditions: freezing, dehydration or anoxia. Purified IDH exhibited higher affinity for isocitrate under all stress conditions as compared to controls, suggesting that the enzyme is primed to synthesize NADPH relative to the control state. Immunoblotting showed reduced serine and threonine phosphorylation of muscle IDH from frozen frogs and decreased serine phosphorylation on IDH from dehydrated frogs relative to control and anoxic states, demonstrating a reversible phosphorylation regulatory mechanism for IDH activity during freezing stress. Taken together, these results suggest activation and maintenance of IDH activity despite hypometabolic conditions. This initiation in activity of IDH during freezing may play a role in antioxidant defense by contributing to maintenance of the NADPH pool under stress conditions.

???displayArticle.pubmedLink??? 36958591
???displayArticle.link??? Biochimie

GO keywords: response to reactive oxygen species [+]

???attribute.lit??? ???displayArticles.show???

	Graphical abstract
	Fig. 1. SDS-PAGE gel showing a representative purification of control muscle IDH from the freeze-tolerant wood frog (R. sylvatica). Lanes are (A) 10–170 kDa protein ladder, (B) crude homogenate, (C) eluate from DEAE+ column, (D) eluate from hydroxyapatite column, and (E) eluate from Cibacron blue column following a 0–2 M KCl gradient.
	Fig. 2. Quantification of post-translational modifications on R. sylvatica skeletal muscle IDH of control, 24 h frozen, 40% dehydrated and 20 h anoxic states. Below the graph are the corresponding (i) ECL bands as determined by chemiluminescence, and (ii) the Coomassie stained bands for each condition. Chemiluminescence signal intensity was standardized to protein amount added, and the control IDH group was standardized to a value of 1. Data are presented as mean ± SEM, n = 4, for purified enzyme samples. ∗ Denotes a significant difference from the corresponding control IDH levels as determined from a Student's t-test, p < 0.05.
	Fig. 3. Assessment of Km isocitrate for cytosolic IDH and the effects of modification by endogenous protein kinases or phosphatases under A) control, B) 24 h frozen, C) 40% dehydrated, and D) 20 h anoxic conditions. Data include the inhibition of all protein kinases and phosphatases (STOP), as well as conditions that promote the activity of total protein kinases (PK Tot), total protein phosphatases (PP Tot), protein kinase A (PKA), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) or serine/threonine phosphatases (PPs). ∗ Denotes a significant difference from the corresponding STOP controls as determined by a Student's t-test, p < 0.05.