Free-radical first responders: The characterization of CuZnSOD and MnSOD regulation during freezing of the freeze-tolerant North American wood frog, Rana sylvatica

• Published in: Biochimica et biophysica acta Vol. 1850; no. 1; pp. 97 - 106
• Main Authors: Dawson, Neal J., Katzenback, Barbara A., Storey, Kenneth B.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2015
• Subjects: Adaptation, Physiological; Amino Acid Sequence; Animals; Antioxidant; blood flow; Blotting, Western; digestion; DNA, Complementary - chemistry; DNA, Complementary - genetics; Enzyme Stability; Free Radicals - metabolism; Freeze tolerance; Freezing; frogs; Gene Expression Regulation, Enzymologic; hydrogen; Ischemia; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Lithobates sylvaticus; Male; messenger RNA; Molecular Sequence Data; muscles; Muscles - enzymology; North America; overwintering; Oxidative stress; oxygen; Phosphorylation; protein phosphorylation; Protein stability; Rana sylvatica; Ranidae - genetics; Ranidae - metabolism; Ranidae - physiology; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology, Amino Acid; serine; superoxide anion; superoxide dismutase; Superoxide Dismutase - genetics; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism; tyrosine; urea; North America; Oxidative stress; Km; DTT; ALS; Protein stability; Cm; Freeze tolerance; NiSOD; CuZnSOD; Ischemia; FeSOD; NBT; IMAC; KPi; Vmax; PKA; TBST; PKC; PP2A; DEPC; MAPK; Antioxidant; IP3; PKG; PVDF; PP1; PP2C; PMSF; MnSOD; ROS; Rana sylvatica; AMPK; DEAE
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2014.10.003

The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O2−) to molecular oxygen and hydrogen peroxide. The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica. CuZnSOD from frozen muscle showed a significantly higher Vmax (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O2− (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (Cm) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53M urea) and frozen (2.92M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different. The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs. Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia. •R. sylvatica freeze over winter, imposing oxidative stress on their cells as a result of ischemia.•sod1 and sod2 partial sequences were obtained from R. sylvatica.•CuZnSOD from frozen frogs shows changes in Vmax, and no change in Km, PTMs or stability.•MnSOD from frozen frogs shows changes in Km, increased phosphorylation and stability.•Freeze-induced changes adjust SOD function to account for rising oxidative stressors.