Skip to main content
SpringerLink
Log in

Purification and characterization of NADP-isocitrate dehydrogenase from skeletal muscle of Urocitellus richardsonii

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

NADP-dependent isocitrate dehydrogenase (NADP-IDH, EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with the concomitant production of NADPH. NADPH plays important roles in many biosynthesis pathways, maintenance of proper oxidation–reduction balance, and protection against oxidative damage. This present study investigated the dynamic nature of NADP-IDH during hibernation by purifying it from the skeletal muscle of Richardson's ground squirrel (Urocitellus richardsonii) and analyzing its structural and functional changes in response to hibernation. Kinetic parameters of purified NADP-IDH from euthermic and hibernating ground squirrel skeletal muscle were characterized at 22 °C and 5 °C. Relative to euthermic muscle, -NADP-IDH in hibernating muscle had a higher affinity for its substrate, isocitrate at 22 °C, whereas at 5 °C, there was a significant decrease in isocitrate affinity. Western blot analysis revealed greater serine and threonine phosphorylation in hibernator NADP-IDH as compared to euthermic NADP-IDH. In addition, Bioinformatic analysis predicted the presence of 18 threonine and 21 serine phosphorylation sites on squirrel NADP-IDH. The structural and functional changes in NADP-IDH indicate the ability of the organism to reduce energy consumption during hibernation, while emphasizing increased NADPH production, and thus antioxidant activity, during torpor arousal cycles.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The data are available on reasonable request.

References

  1. Lyman CP, Willis JS, Malan A, Wang LCH (1984) Hibernation and torpor in mammals and birds. Academic Press, New York

    Google Scholar 

  2. Abnous K, Dieni CA, Storey KB (2008) Regulation of Akt during hibernation in Richardson’s ground squirrels. Biochim Biophys Acta—Gen Subj 1780:185–193. https://doi.org/10.1016/j.bbagen.2007.10.009

    Article  CAS  Google Scholar 

  3. Ruberto AA, Childers CL, Storey KB (2016) Purification and properties of glycerol-3-phosphate dehydrogenase from the liver of the hibernating ground squirrel, Urocitellus richardsonii. Comp Biochem Physiol Part B Biochem Mol Biol 202:48–55. https://doi.org/10.1016/j.cbpb.2016.08.001

    Article  CAS  Google Scholar 

  4. Wang LCH (1979) Time patterns and metabolic rates of natural torpor in the Richardson’s ground squirrel. Can J Zool 57:149–155. https://doi.org/10.1139/z79-012

    Article  Google Scholar 

  5. Landau BR, Dawe AR (1958) Respiration in the hibernation of the 13-lined ground squirrel. Am J Physiol Content 194:75–82. https://doi.org/10.1152/ajplegacy.1958.194.1.75

    Article  CAS  Google Scholar 

  6. Buck CL, Barnes BM (2000) Effects of ambient temperature on metabolic rate, respiratory quotient, and torpor in an arctic hibernator. Am J Physiol Regul Integr Comp Physiol 279:R255–R262. https://doi.org/10.1152/ajpregu.2000.279.1.R255

    Article  CAS  Google Scholar 

  7. Wang LCH, Lee TF (1996) Torpor and hibernation in mammals: metabolic, physiological, and biochemical adaptations. Comprehensive Physiology. Wiley, Hoboken, pp 507–532

    Chapter  Google Scholar 

  8. Storey KB (1997) Metabolic regulation in mammalian hibernation: enzyme and protein adaptations. Comp Biochem Physiol A Physiol 118:1115–1124. https://doi.org/10.1016/s0300-9629(97)00238-7

    Article  CAS  Google Scholar 

  9. Carey HV, Andrews MT, Martin SL (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83:1153–1181. https://doi.org/10.1152/physrev.00008.2003

    Article  CAS  Google Scholar 

  10. Storey KB, Storey JM (2004) Metabolic rate depression in animals: transcriptional and translational controls. Biol Rev Camb Philos Soc 79:207–233. https://doi.org/10.1017/s1464793103006195

    Article  Google Scholar 

  11. Brooks SP, Storey KB (1992) Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis. J Comp Physiol B 162:23–28. https://doi.org/10.1007/BF00257932

    Article  CAS  Google Scholar 

  12. MacDonald JA, Storey KB (1998) cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels. J Comp Physiol B 168:513–525. https://doi.org/10.1007/s003600050172

    Article  CAS  Google Scholar 

  13. Xu X, Zhao J, Xu Z et al (2004) Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. J Biol Chem 279:33946–33957. https://doi.org/10.1074/jbc.M404298200

    Article  CAS  Google Scholar 

  14. Ju H-Q, Lin J-F, Tian T et al (2020) NADPH homeostasis in cancer: functions, mechanisms and therapeutic implications. Signal Transduct Target Ther 5:231. https://doi.org/10.1038/s41392-020-00326-0

    Article  CAS  Google Scholar 

  15. Thatcher BJ, Storey KB (2001) Glutamate dehydrogenase from liver of euthermic and hibernating Richardson’s ground squirrels: evidence for two distinct enzyme forms. Biochem Cell Biol 79:11–19. https://doi.org/10.1139/o00-086

    Article  CAS  Google Scholar 

  16. Bell RAV, Dawson NJ, Storey KB (2012) Insights into the in vivo regulation of glutamate dehydrogenase from the foot muscle of an estivating land snail. Enzyme Res 2012:1–10. https://doi.org/10.1155/2012/317314

    Article  CAS  Google Scholar 

  17. 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 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  Google Scholar 

  18. Brooks SPJ (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13(6):906–911

    CAS  Google Scholar 

  19. Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362. https://doi.org/10.1006/jmbi.1999.3310

    Article  CAS  Google Scholar 

  20. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. https://doi.org/10.1093/bioinformatics/bti770

    Article  CAS  Google Scholar 

  21. Bordoli L, Kiefer F, Arnold K et al (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4:1–13. https://doi.org/10.1038/nprot.2008.197

    Article  CAS  Google Scholar 

  22. Biasini M, Bienert S, Waterhouse A et al (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258. https://doi.org/10.1093/nar/gku340

    Article  CAS  Google Scholar 

  23. Brooks SPJ (1994) A program for analyzing enzyme rate data obtained from a microplate reader. Biotechniques 17(6):1154–1161

    CAS  Google Scholar 

  24. Zhang J, Storey KB (2016) RBioplot: an easy-to-use R pipeline for automated statistical analysis and data visualization in molecular biology and biochemistry. PeerJ 4:e2436. https://doi.org/10.7717/peerj.2436

    Article  Google Scholar 

  25. Storey KB (2003) Mammalian hibernation. Transcriptional and translational controls. Adv Exp Med Biol 543:21–38

    Article  CAS  Google Scholar 

  26. Ruf T, Geiser F (2015) Daily torpor and hibernation in birds and mammals. Biol Rev 90:891–926. https://doi.org/10.1111/brv.12137

    Article  Google Scholar 

  27. Orr AL, Lohse LA, Drew KL, Hermes-Lima M (2009) Physiological oxidative stress after arousal from hibernation in arctic ground squirrel. Comp Biochem Physiol A Mol Integr Physiol 153:213–221. https://doi.org/10.1016/j.cbpa.2009.02.016

    Article  CAS  Google Scholar 

  28. Storey KB, Storey JM (2012) Aestivation: signaling and hypometabolism. J Exp Biol 215:1425–1433. https://doi.org/10.1242/jeb.054403

    Article  CAS  Google Scholar 

  29. Krivoruchko A, Storey KB (2015) Turtle anoxia tolerance: biochemistry and gene regulation. Biochim Biophys Acta—Gen Subj 1850:1188–1196. https://doi.org/10.1016/j.bbagen.2015.02.001

    Article  CAS  Google Scholar 

  30. Moreira DC, Venancio LPR, Sabino MACT, Hermes-Lima M (2016) How widespread is preparation for oxidative stress in the animal kingdom? Comp Biochem Physiol Part A Mol Integr Physiol 200:64–78. https://doi.org/10.1016/j.cbpa.2016.01.023

    Article  CAS  Google Scholar 

  31. Tessier SN, Breedon SA, Storey KB (2021) Modulating Nrf2 transcription factor activity: revealing the regulatory mechanisms of antioxidant defenses during hibernation in 13-lined ground squirrels. Cell Biochem Funct 39:623–635. https://doi.org/10.1002/cbf.3627

    Article  CAS  Google Scholar 

  32. Zera AJ, Newman S, Berkheim D et al (2011) Purification and characterization of cytoplasmic NADP+-isocitrate dehydrogenase, and amplification of the NADP+-IDH gene from the wing-dimorphic sand field cricket Gryllus firmus. J Insect Sci 11:53. https://doi.org/10.1673/031.011.5301

    Article  Google Scholar 

  33. Vucetic M, Stancic A, Otasevic V et al (2013) The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus). Free Radic Biol Med 65:916–924. https://doi.org/10.1016/j.freeradbiomed.2013.08.188

    Article  CAS  Google Scholar 

  34. Allan ME, Storey KB (2012) Expression of NF-κB and downstream antioxidant genes in skeletal muscle of hibernating ground squirrels, Spermophilus tridecemlineatus. Cell Biochem Funct 30:166–174. https://doi.org/10.1002/cbf.1832

    Article  CAS  Google Scholar 

  35. Morin P, Storey KB (2006) Evidence for a reduced transcriptional state during hibernation in ground squirrels. Cryobiology 53:310–318. https://doi.org/10.1016/j.cryobiol.2006.08.002

    Article  CAS  Google Scholar 

  36. Frerichs KU, Smith CB, Brenner M et al (1998) Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation. Proc Natl Acad Sci 95:14511–14516. https://doi.org/10.1073/pnas.95.24.14511

    Article  CAS  Google Scholar 

  37. Cai D, McCarron RM, Yu EZ et al (2004) Akt phosphorylation and kinase activity are down-regulated during hibernation in the 13-lined ground squirrel. Brain Res 1014:14–21. https://doi.org/10.1016/j.brainres.2004.04.008

    Article  CAS  Google Scholar 

  38. Storey KB (2010) Out cold: biochemical regulation of mammalian hibernation—A mini-review. Gerontology 56:220–230. https://doi.org/10.1159/000228829

    Article  Google Scholar 

  39. Biggar Y, Storey KB (2014) Global DNA modifications suppress transcription in brown adipose tissue during hibernation. Cryobiology 69:333–338. https://doi.org/10.1016/j.cryobiol.2014.08.008

    Article  CAS  Google Scholar 

  40. Brown JCL, Chung DJ, Belgrave KR, Staples JF (2012) Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels. Am J Physiol Regul Integr Comp Physiol 302:R15-28. https://doi.org/10.1152/ajpregu.00230.2011

    Article  CAS  Google Scholar 

  41. Abnous K, Storey KB (2007) Regulation of skeletal muscle creatine kinase from a hibernating mammal. Arch Biochem Biophys 467:10–19. https://doi.org/10.1016/j.abb.2007.07.025

    Article  CAS  Google Scholar 

  42. Abnous K, Storey KB (2008) Skeletal muscle hexokinase: regulation in mammalian hibernation. Mol Cell Biochem 319:41–50. https://doi.org/10.1007/s11010-008-9875-5

    Article  CAS  Google Scholar 

  43. 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 442:47–58. https://doi.org/10.1007/s11010-017-3192-9

    Article  CAS  Google Scholar 

  44. Green SR, Storey KB (2021) Skeletal muscle of torpid Richardson’s ground squirrels (Urocitellus richardsonii) exhibits a less active form of citrate synthase associated with lowered lysine succinylation. Cryobiology 101:28–37. https://doi.org/10.1016/j.cryobiol.2021.06.006

    Article  CAS  Google Scholar 

  45. Ramnanan CJ, McMullen DC, Bielecki A, Storey KB (2010) Regulation of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) in turtle muscle and liver during acute exposure to anoxia. J Exp Biol 213:17–25. https://doi.org/10.1242/jeb.036087

    Article  CAS  Google Scholar 

  46. Ruberto AA, Logan SM, Storey KB (2019) Temperature and serine phosphorylation regulate glycerol-3-phosphate dehydrogenase in skeletal muscle of hibernating Richardson’s ground squirrels. Biochem Cell Biol 97:148–157. https://doi.org/10.1139/bcb-2018-0198

    Article  CAS  Google Scholar 

  47. Storey KB, Storey JM (2005) Biochemical adaptation to extreme environments. Integrative Physiology in the Proteomics and Post-Genomics Age. Humana Press, Totowa, pp 169–200

    Chapter  Google Scholar 

Download references

Acknowledgements

The authors thank S.A. Breedon and C.L. Childers for their help and editorial review of this manuscript. The authors also thank J.M. Storey for editorial review of this manuscript.

Funding

The research was funded by a Discovery Grant (Number# 6793) through the Natural Sciences and Engineering Research Council of Canada (NSERC). K.B. Storey currently holds the Canada Research Chair in Molecular Physiology.

Author information

Author notes

  1. Isabelle A. MacLean and Anchal Varma have contributed equally to this work.

Authors and Affiliations

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

    Isabelle A. MacLean, Anchal Varma & Kenneth B. Storey

Authors
  1. Isabelle A. MacLean
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anchal Varma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kenneth B. Storey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

IAM and KBS conceptualized and designed the project and IAM conducted all experiments. Data analysis and assembly of the manuscript were carried out by AV, IAM, and KBS.

Corresponding author

Correspondence to Kenneth B. Storey.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All protocols for animal care and treatment had prior approval by the Carleton University Animal Care Committee and met guidelines for the Canadian Council on Animal Care.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

MacLean, I.A., Varma, A. & Storey, K.B. Purification and characterization of NADP-isocitrate dehydrogenase from skeletal muscle of Urocitellus richardsonii. Mol Cell Biochem 478, 415–426 (2023). https://doi.org/10.1007/s11010-022-04516-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-022-04516-y

Keywords

Search

Navigation