Purification and characterization of a urea sensitive lactate dehydrogenase from skeletal muscle of the African clawed frog, Xenopus laevis

  • Christine L. Childers
  • Kenneth B. StoreyEmail author
Original Paper


The African clawed frog, Xenopus laevis endures whole body dehydration which can increase its reliance on anaerobic glycolysis for energy production. This makes the regulation of the terminal enzyme of glycolysis, lactate dehydrogenase (LDH), crucial to stress survival. We investigated the enzymatic properties and posttranslational modification state of purified LDH from the skeletal muscle of control and dehydrated (30% total body water loss) X. laevis. LDH from the muscle of dehydrated frogs showed a 93% reduction in phosphorylation on threonine residues and an 80% reduction of protein nitrosylation. LDH from dehydrated muscle also showed a 74% lower Vmax in the pyruvate oxidizing direction and a 78% decrease in Vmax in the lactate reducing direction along with a 33% lower Km for pyruvate and a 40% higher Km for lactate. In the presence of higher levels of urea and molecular crowding by polyethylene glycol, used to mimic conditions in the cells of dehydrated animals, the Km values of control and dehydrated LDH demonstrated opposite responses. In the pyruvate oxidizing direction, control muscle LDH was unaffected by these additives, whereas the affinity for pyruvate dropped further for LDH from dehydrated muscle. The opposite effect was more pronounced in the lactate reducing direction as control LDH showed an increased affinity for lactate, whereas LDH from dehydrated animals showed a further reduction in affinity. The physiological consequences of dehydration-induced LDH regulation appear to poise the enzyme towards lactate production when urea levels are high and lactate catabolism when urea levels are low, perhaps helping to maintain glycolysis under dehydrating conditions whilst providing for the ability to recycle lactate upon rehydration.


Lactate dehydrogenase Xenopus laevis Dehydration Urea 



We thank J. M. Storey for critical commentary on the manuscript. This work was supported by a Discovery Grant (#6793) from the Natural Sciences and Engineering Research Council of Canada; KBS holds the Canada Research Chair in Molecular Physiology.


  1. Abboud J, Storey KB (2013) Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications. PeerJ 1:e12. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alexander SS, Bellerby CW (1938) Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis). I. J Exp Biol 15:74–81Google Scholar
  3. Balinsky JB, Choritz EL, Coe CG, van der Schans GS (1967a) Amino acid metabolism and urea synthesis in naturally aestivating Xenopus laevis. Comp Biochem Physiol 22:59–68CrossRefGoogle Scholar
  4. Balinsky JB, Choritz EL, Coe CGL, van der Schans GS (1967b) Amino acid metabolism and urea synthesis in naturally aestivating Xenopus laevis. Comp Biochem Physiol 22:59–68. CrossRefPubMedGoogle Scholar
  5. Bellerby CW (1938) Experimental studies on the sexual cycle of the South African Clawed Toad (Xenopus laevis). II. J exp Biol London 15:82–90Google Scholar
  6. Biggar K, Dawson N, Storey K (2012) Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler. Biotechniques 53:231–238. CrossRefPubMedGoogle Scholar
  7. Brooks SP (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13(6):906–911PubMedGoogle Scholar
  8. Childers CL, Storey KB (2016) Post-translational regulation of hexokinase function and protein stability in the aestivating frog Xenopus laevis. Protein J 35:61–71. CrossRefPubMedGoogle Scholar
  9. Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol 4:E127–E130. CrossRefPubMedGoogle Scholar
  10. Dawson NJ, Bell RAV, Storey KB (2013) Purification and properties of white muscle lactate dehydrogenase from the anoxia-tolerant turtle, the red-eared slider, Trachemys scripta elegans. Enzyme Res 2013:784973. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Eggert C, Fouquet A (2006) A preliminary biotelemetric study of a feral invasive Xenopus laevis population in France. Alytes 23:144–149Google Scholar
  12. Flanigan JE, WIthers PC, Guppy M (1991) In vitro metabolic depression of tissues from the aestivating frog Neobatrachus pelobatoides. J Exp Biol 161Google Scholar
  13. Gatten RE (1987) Activity metabolism of anuran amphibians: tolerance to dehydration. Physiol Zool 60:576–585. CrossRefGoogle Scholar
  14. Guppy M, Withers P (1999) Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol Rev 74:1–40CrossRefGoogle Scholar
  15. Hillman SS (1978) The roles of oxygen delivery and electrolyte levels in the dehydrational death of Xenopus laevis. J Comp Physiol B 128:169–175. CrossRefGoogle Scholar
  16. Hillman SS, Sommerfeldt RW (1981) Microsphere studies of amphibian systemic blood flow redistribution during dehydration, hypovolemia, and salt load. J Exp Zool 218:305–308. CrossRefGoogle Scholar
  17. Hochachka PW, Somero GN (1984) Biochemical adaptation. Princeton University Press, PrincetonCrossRefGoogle Scholar
  18. Katzenback BA, Dawson NJ, Storey KB (2014) Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis. J Comp Physiol B 184:601–611. CrossRefPubMedGoogle Scholar
  19. MacDonald JA, Storey KB (1999) Regulation of ground squirrel Na+K+-ATPase activity by reversible phosphorylation during hibernation. Biochem Biophys Res Commun 254:424–429. CrossRefPubMedGoogle Scholar
  20. MacLean IA, Mattice AMS, Adam NJ, Storey KB (2016) Purification and characterization of lactate dehydrogenase in the foot muscle and hepatopancreas of Otala lactea. Protein J 35:467–480. CrossRefPubMedGoogle Scholar
  21. Malik AI, Storey KB (2009) Activation of extracellular signal-regulated kinases during dehydration in the African clawed frog, Xenopus laevis. J Exp Biol 212:2595–2603. CrossRefPubMedGoogle Scholar
  22. Mashino T, Fridovich I (1987) Effects of urea and trimethylamine-N-oxide on enzyme activity and stability. Arch Biochem Biophys 258:356–360. CrossRefPubMedGoogle Scholar
  23. Measey G, Tinsley R (1998) Feral Xenopus laevis in south Wales. Herpetol J 8:23–28Google Scholar
  24. Muir TJ, Costanzo JP, Lee RE (2007) Osmotic and metabolic responses to dehydration and urea-loading in a dormant, terrestrially hibernating frog. J Comp Physiol B Biochem Syst Environ Physiol. CrossRefGoogle Scholar
  25. Müller MM (2018) Post-translational modifications of protein backbones: unique functions, mechanisms, and challenges. Biochemistry 57:177–185. CrossRefPubMedGoogle Scholar
  26. Oliveira AP, Sauer U (2012) The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism. FEMS Yeast Res 12:104–117. CrossRefPubMedGoogle Scholar
  27. Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131:266–280. CrossRefPubMedGoogle Scholar
  28. Shahriari A, Dawson NJ, Bell RAV, Storey KB (2013) Stable suppression of lactate dehydrogenase activity during anoxia in the foot muscle of Littorina littorea and the potential role of acetylation as a novel posttranslational regulatory mechanism. Enzyme Res 2013:461374. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Storey KB (2016) Comparative enzymology—new insights from studies of an “old” enzyme, lactate dehydrogenase. Comp Biochem Physiol Part B Biochem Mol Biol 199:13–20. CrossRefGoogle Scholar
  30. Storey KB, Storey JM (1990) Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation. Q Rev Biol 65:145–174CrossRefGoogle Scholar
  31. Storey KB, Storey JM (2005) Oxygen limitation and metabolic rate depression. In: Storey KB (ed) Functional metabolism. Wiley, Hoboken, pp 415–442CrossRefGoogle Scholar
  32. Storey KB, Storey JM (2012) Aestivation: signaling and hypometabolism. J Exp Biol 215:1425–1433. CrossRefPubMedGoogle Scholar
  33. Talaiezadeh A, Shahriari A, Tabandeh MR et al (2015) Kinetic characterization of lactate dehydrogenase in normal and malignant human breast tissues. Cancer Cell Int 15:19. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Uchiyama M, Konno N (2006) Hormonal regulation of ion and water transport in anuran amphibians. Gen Comp Endocrinol 147:54–61. CrossRefPubMedGoogle Scholar
  35. Walsh CT, Garneau-Tsodikova S, Gatto GJ (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed 44:7342–7372. CrossRefGoogle Scholar
  36. Xiong ZJ, Storey KB (2012) Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle. Comp Biochem Physiol Part B Biochem Mol Biol 163:221–228. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology, Institute of BiochemistryCarleton UniversityOttawaCanada
  2. 2.Department of Biology and Chemistry, Institute of BiochemistryCarleton UniversityOttawaCanada

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