title: Free-radical first responders: the characterization of CuZnSOD and MnSOD regulation during freezing of the freeze-tolerant North American wood frog, Rana sylvatica.
abstract: BACKGROUND
  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 (O(2)(-)) to molecular oxygen and hydrogen peroxide.
  
  
  METHODS
  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.
  
  
  RESULTS
  CuZnSOD from frozen muscle showed a significantly higher V(max) (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O(2)(-) (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 (C(m)) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different.
  
  
  CONCLUSIONS
  The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs.
  
  
  CONCLUSIONS
  Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.
authors.affiliation: Department of Biology and Institute of Biochemistry Carleton University, Ottawa, ON, Canada.
authors.forename: Neal J
authors.initials: NJ
authors.lastname: Dawson
authors.affiliation: Department of Biology and Institute of Biochemistry Carleton University, Ottawa, ON, Canada.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Katzenback
authors.affiliation: Department of Biology and Institute of Biochemistry Carleton University, Ottawa, ON, Canada. Electronic address: kenneth_storey@carleton.ca.
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Storey
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005609:Free Radicals / Q000378:metabolism*; D005615:Freezing; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009656:North America; D010766:Phosphorylation; D011898:Ranidae / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D005609:Free Radicals; D007527:Isoenzymes; D013482:Superoxide Dismutase
keywords: Antioxidant; Freeze tolerance; Ischemia; Oxidative stress; Protein stability; Rana sylvatica