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Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea

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Abstract

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

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Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Lutz PL, Storey KB (2011) Adaptations to variations in oxygen tension by vertebrates and invertebrates. Compr Physiol. https://doi.org/10.1002/cphy.cp130221

    Article  Google Scholar 

  2. De Zwaan A, Putzer V (1985) Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia). Symp Soc Exp Biol 39:33–62

    PubMed  Google Scholar 

  3. Aarset AV (1982) Freezing tolerance in intertidal invertebrates (a review). Comp Biochem Physiol -Part A Physiol. https://doi.org/10.1016/0300-9629(82)90264-X

    Article  Google Scholar 

  4. English TE, Storey KB (2003) Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea. J Exp Biol. https://doi.org/10.1242/jeb.00465

    Article  PubMed  Google Scholar 

  5. Murphy DJ (1983) Freezing Resistance in Intertidal Invertebrates. Annu Rev Physiol. https://doi.org/10.1146/annurev.ph.45.030183.001445

    Article  PubMed  Google Scholar 

  6. Larade K, Storey KB (2002) Reversible suppression of protein synthesis in concert with polysome disaggregation during anoxia exposure in Littorina littorea. Mol Cell Biochem. https://doi.org/10.1023/A:1014811017753

    Article  PubMed  Google Scholar 

  7. Larade K, Storey KB (2007) Arrest of transcription following anoxic exposure in a marine mollusc. Mol Cell Biochem. https://doi.org/10.1007/s11010-007-9468-8

    Article  PubMed  Google Scholar 

  8. Biggar KK, Kornfeld SF, Maistrovski Y, Storey KB (2012) MicroRNA regulation in extreme environments: differential expression of MicroRNAs in the intertidal snail Littorina littorea during extended periods of freezing and anoxia. Genomics Proteomics Bioinforma. https://doi.org/10.1016/j.gpb.2012.09.002

    Article  Google Scholar 

  9. Storey KB, Lant B, Anozie OO, Storey JM (2013) Metabolic mechanisms for anoxia tolerance and freezing survival in the intertidal gastropod Littorina littorea. Comp Biochem Physiol. https://doi.org/10.1016/j.cbpa.2013.03.00

    Article  Google Scholar 

  10. Krivoruchko A, Storey KB (2015) Turtle anoxia tolerance: biochemistry and gene regulation. Biochim Biophys Acta. https://doi.org/10.1016/j.bbagen.2015.02.001

    Article  PubMed  Google Scholar 

  11. Hand SC, Hardewig I (1996) Downregulation of cellular metabolism during environmental stress: mechanisms and implications. Annu Rev Physiol. https://doi.org/10.1146/annurev.ph.58.030196.002543

    Article  PubMed  Google Scholar 

  12. Brooks SPJ, Storey KB (1997) Glycolytic controls in estivation and anoxia: a comparison of metabolic arrest in land and marine molluscs. Comp Biochem Physiol 118:1103–1114

    Article  CAS  Google Scholar 

  13. Russell EL, Storey KB (1995) Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea. J Comp Physiol B. https://doi.org/10.1007/BF00301477

    Article  Google Scholar 

  14. Lama JL, Bell RAV, Storey KB (2013) Hexokinase regulation in the hepatopancreas and foot muscle of the anoxia-tolerant marine mollusk. Comp Biochem Physiol. https://doi.org/10.1016/j.cbpb.2013.07.001

    Article  Google Scholar 

  15. 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. https://doi.org/10.1155/2013/461374

    Article  PubMed  PubMed Central  Google Scholar 

  16. Argaud D, Roth H, Wiernsperger N, Leverve XM (1993) Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes. Eur J Biochem. https://doi.org/10.1111/j.1432-1033.1993.tb17886.x

    Article  PubMed  Google Scholar 

  17. Rognstad R, Katz J (1977) Role of pyruvate kinase in the regulation of gluconeogenesis from L lactate. J Biol Chem 252:1831–1833

    CAS  PubMed  Google Scholar 

  18. Plaxton WC, Storey KB (1984) Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channelled whelk, Busycotypus canaliculatum. Eur J Biochem. https://doi.org/10.1111/j.1432-1033.1984.tb08367.x

    Article  PubMed  Google Scholar 

  19. Plaxton WC, Storey KB (1985) Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum. Arch Biochem Biophys. https://doi.org/10.1016/0003-9861(85)90788-X

    Article  PubMed  Google Scholar 

  20. Whitwam RE, Storey KS (1990) Pyruvate kinase from the land snail Otala lactea: regulation by reversible phosphorylation during estivation and anoxia. J Exp Biol 154:321–337

    CAS  Google Scholar 

  21. Smolinski MB, Mattice JJL, Storey KB (2017) Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog, Rana sylvatica. Cryobiology. https://doi.org/10.1016/j.cryobiol.2017.06.002

    Article  PubMed  Google Scholar 

  22. Mattice AMS, MacLean IA, Childers CL, Storey KB (2018) Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression. PeerJ. https://doi.org/10.7717/peerj.4918

    Article  PubMed  PubMed Central  Google Scholar 

  23. Bell RAV, Storey KB (2018) Purification and characterization of skeletal muscle pyruvate kinase from the hibernating ground squirrel, Urocitellus richardsonii: potential regulation by posttranslational modification during torpor. Mol Cell Biochem. https://doi.org/10.1007/s11010-017-3192-9

    Article  PubMed  Google Scholar 

  24. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  PubMed  Google Scholar 

  25. Brooks SPJ (1994) A program for analyzing enzyme rate data obtained from a microplate reader. Biotechniques 17:1154–1161

    CAS  PubMed  Google Scholar 

  26. Brooks SPJ (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13:906–911

    CAS  PubMed  Google Scholar 

  27. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal omega. Mol Syst Biol. https://doi.org/10.1038/msb.2011.75

    Article  PubMed  PubMed Central  Google Scholar 

  28. Blom N, Sicheritz-Pontén T, Gupta R, Gammeltoft S, Brunak S (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics. https://doi.org/10.1002/pmic.200300771

    Article  PubMed  Google Scholar 

  29. Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V, Sullivan M (2012) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. https://doi.org/10.1093/nar/gkr1122

    Article  PubMed  Google Scholar 

  30. Storey KB, Storey JM (1990) Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation. Q Rev Biol. https://doi.org/10.1086/416717

    Article  PubMed  Google Scholar 

  31. Storey KB (1997) Metabolic regulation in mammalian hibernation: Enzyme and protein adaptations. Comp Biochem Physiol. https://doi.org/10.1016/S0300-9629(97)00238-7

    Article  Google Scholar 

  32. Cowan KJ, Storey KB (1999) Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii. Mol Cell Biochem. https://doi.org/10.1023/A:1006932221288

    Article  PubMed  Google Scholar 

  33. Storey KB, Storey JM (2004) Oxygen limitation and metabolic rate depression. In: Storey KB (ed) Functional metabolism, 1st edn. Wiley, Hoboken, pp 415–442

    Chapter  Google Scholar 

  34. Churchill TA, Storey KB (1996) Metabolic responses to freezing and anoxia by the periwinkle Littorina littorea. J Therm Biol. https://doi.org/10.1016/0306-4565(95)00022-4

    Article  Google Scholar 

  35. Wieser W (1980) Metabolic end products in three species of marine gastropods. J Mar Biol Assoc UK. https://doi.org/10.1017/S0025315400024231

    Article  Google Scholar 

  36. Storey KB (1986) Aspartate activation of pyruvate kinase in anoxia tolerant molluscs. Comp Biochem Physiol. https://doi.org/10.1016/0305-0491(86)90151-3

    Article  Google Scholar 

  37. Sokolova IM, Pörtner HO (2001) Temperature effects on key metabolic enzymes in Littorina saxatilis and Littorina obtusata from different latitudes and shore levels. Mar Biol. https://doi.org/10.1007/s002270100557

    Article  Google Scholar 

  38. Sokolova IM, Bock C, Pörtner HO (2000) Resistance to freshwater exposure in White Sea Littorina spp. I: anaerobic metabolism and energetics. J Comp Physiol. https://doi.org/10.1007/s003600050264

    Article  Google Scholar 

  39. Hoyaux J, Gilles R, Jeuniaux C (1976) Osmoregulation in molluscs of the intertidal zone. Comp Biochem Physiol Part A. https://doi.org/10.1016/S0300-9629(76)80157-0

    Article  Google Scholar 

  40. Taylor PM, Andrews EB (1988) Osmoregulation in the intertidal gastropod Littorina littorea. J Exp Mar Bio Ecol. https://doi.org/10.1016/0022-0981(88)90210-9

    Article  Google Scholar 

  41. Pannunzio TM, Storey KB (1998) Antioxidant defenses and lipid peroxidation during anoxia stress and aerobic recovery in the marine gastropod Littorina littorea. J Exp Mar Bio Ecol. https://doi.org/10.1016/S0022-0981(97)00132-9

    Article  Google Scholar 

  42. Storey KB, Storey JM (1988) Freeze tolerance in animals. Physiol Rev. https://doi.org/10.1152/physrev.1988.68.1.27

    Article  PubMed  Google Scholar 

  43. Abboud J, Storey KB (2013) Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications. PeerJ. https://doi.org/10.7717/peerj.12

    Article  PubMed  PubMed Central  Google Scholar 

  44. Titanji VPK, Zetterqvist Ö, Engström L (1976) Regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions, catalyzed by cyclic-AMP dependent protein kinases and a histone phosphatase. Biochim Biophys Acta. https://doi.org/10.1016/0005-2744(76)90011-5

    Article  PubMed  Google Scholar 

  45. Ljungström O, Berglund L, Engström L (1976) Studies on the kinetic effects of adenosine-3′: 5′-monophosphatedependent phosphorylation of purified pig-liver pyruvate kinase type L. Eur J Biochem. https://doi.org/10.1111/j.1432-1033.1976.tb10837.x

    Article  Google Scholar 

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Acknowledgements

This study was funded through a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (#6793). K.B.S. holds the Canada Research Chair in Molecular Physiology. M.B.S was funded through an Ontario Graduate Scholarship. The authors thank J.M. Storey for editorial review of this manuscript.

Funding

This study was funded through a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (#6793). K.B.S. holds the Canada Research Chair in Molecular Physiology. M.B.S was funded through an Ontario Graduate Scholarship.

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  1. Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada

    Michael B. Smolinski, Anchal Varma, Stuart R. Green & Kenneth B. Storey

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  1. Michael B. Smolinski
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  2. Anchal Varma
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  4. Kenneth B. Storey
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Contributions

MBS carried out experiments, analyzed the data, and helped draft the manuscript. AV analyzed the data and drafted the manuscript. SRG carried out experiments and revised the manuscript. KBS secured funding, conceived the experiments, and helped draft the manuscript. All authors read and approved the final manuscript.

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Correspondence to Kenneth B. Storey.

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Protocols were approved by the Carleton University Animal Care Committee.

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Smolinski, M.B., Varma, A., Green, S.R. et al. Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea. Protein J 39, 531–541 (2020). https://doi.org/10.1007/s10930-020-09934-9

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