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The Protein Journal

, Volume 27, Issue 1, pp 43–49 | Cite as

Cytoplasmic and Mitochondrial Creatine Kinases from the Skeletal Muscle of Sperm Whale (Physeter macrocephalus). Molecular Cloning and Enzyme Characterization

  • Kentaro Iwanami
  • Kouji Uda
  • Hiroshi Tada
  • Tomohiko Suzuki
Article
  • 101 Downloads

Abstract

We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of sperm whale MCK and sMiCK showed 94–96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods (neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K m, K d and k cat) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that sperm whale MCK has a comparable affinity for creatine (K m Cr  = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the MCK and sMiCK of sperm whale display a synergistic substrate binding (K d /K m = 3.1–7.8) like those of other mammalian CKs.

Keywords

Creatine kinase Mitochondrial Phosphagen kinase Guanidino kinase Kinetic constant Cetacean Sperm whale Physeter macrocephalus 

Abbreviations

AK

arginine kinase

CK

creatine kinase

MCK

muscle-type isoform of CK

BCK

brain-type isoform of CK

SmiCK

sarcomeric mitochondrial isoform of CK

UmiCK

ubiquitous mitochondrial isoform of CK

Cr

creatine; MBP, maltose-binding protein

Notes

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research in Japan to TS (17570062).

References

  1. 1.
    Ellington WR (2001) Ann Rev Physiol 63:289–325CrossRefGoogle Scholar
  2. 2.
    Wyss M, Smeitink J, Wevers RA, Wallimann T (1992) Biochim Biophys Acta 1102:119–166CrossRefGoogle Scholar
  3. 3.
    McLeish MJ, Kenyon GL (2005) Crit Rev Biochem Mol Biol 40:1–20CrossRefGoogle Scholar
  4. 4.
    Schlattner U, Tokarska-Schlattner M, Wallimann T (2005) Biochim Biophys Acta 1762:164–180 Google Scholar
  5. 5.
    Ellington WR, and Suzuki T (2006) In: Vial C (ed) Molecular anatomy and physiology of proteins – creatine kinase. New York, NovaScience, p 1Google Scholar
  6. 6.
    Suzuki T, Mizuta C, Uda K, Ishida K, Mizuta K, Sona S, Compaan DM, Ellington WR (2004) J Mol Evol 59:218–226CrossRefGoogle Scholar
  7. 7.
    Sona S, Suzuki T, Ellington WR (2004) Biochem Biophys Res Commun 317:1207–1214CrossRefGoogle Scholar
  8. 8.
    Qin W, Khuchua Z, Cheng J, Boero J, Payne RM, Strauss AW (1998) Molec Cell Biochem 184:153–167CrossRefGoogle Scholar
  9. 9.
    Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM (1992) Biochem J 281:21–40Google Scholar
  10. 10.
    Noren SR, Williams TM (2000) Comp Biochem Physiol A Mol Integr Physiol 126:181–191CrossRefGoogle Scholar
  11. 11.
    Dolar ML, Suarez P, Ponganis PJ, Kooyman GL (1999) J Exp Biol 202:227–236 Google Scholar
  12. 12.
    Suzuki T, Furukohri T (1994) J Mol Biol 237:353–357CrossRefGoogle Scholar
  13. 13.
    Borson ND, Salo WL, Drewes LR (1992) PCR Methods Appl 2:144–148Google Scholar
  14. 14.
    Huelsenbeck JP, Ronquist F (2001) Bioinformatics 17:754–755CrossRefGoogle Scholar
  15. 15.
    Uda K, Saishoji N, Ichinari S, Ellington WR, Suzuki T (2005) FEBS J 272:3521–3530CrossRefGoogle Scholar
  16. 16.
    Suzuki T, Tomoyuki T, Uda K (2003) FEBS Lett 533:95–98CrossRefGoogle Scholar
  17. 17.
    Fujimoto N, Tanaka K, Suzuki T (2005) FEBS Lett 579:1688–1692CrossRefGoogle Scholar
  18. 18.
    Morrison JF, James E (1965) Biochem J 97:37–52Google Scholar
  19. 19.
    Cleland WW (1979) Methods Enzymol 63:103–138CrossRefGoogle Scholar
  20. 20.
    Lahiri SD, Wang PF, Babbitt PC, McLeish MJ, Kenyon GL, Allen KN (2002) Biochemistry 41:13861–13867 CrossRefGoogle Scholar
  21. 21.
    May-Collado L, Agnarsson I (2006) Mol Phylogenet Evol 38: 344–354CrossRefGoogle Scholar
  22. 22.
    Suzuki T, Yamamoto Y, Umekawa M (2000b) Biochem J 351:579–585CrossRefGoogle Scholar
  23. 23.
    Takeuchi M, Mizuta C, Uda K, Fujimoto N, Okamoto M, Suzuki T (2004) Cell Mol Life Sci 61:110–117CrossRefGoogle Scholar
  24. 24.
    Matsushima K, Uda K, Ishida K, Kokufuta C, Iwasaki N, Suzuki T (2006) Int J Biol Macromol 38:83–88 CrossRefGoogle Scholar
  25. 25.
    Novak WR, Wang PF, McLeish MJ, Kenyon GL, Babbitt PC (2004) Biochemistry 43:13766–13774CrossRefGoogle Scholar
  26. 26.
    Chen LH, White CB, Babbitt PC, McLeish MJ, Kenyon GL (2000) J Protein Chem 19:59–66CrossRefGoogle Scholar
  27. 27.
    Schlattner U, Eder M, Dolder M, Khuchua ZA, Strauss AW, Wallimann T (2000) Biol Chem 381:1063–1070CrossRefGoogle Scholar
  28. 28.
    Suzuki T, Fukuta H, Nagato H, Umekawa M (2000) J Biol Chem 275:23884–23890CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Kentaro Iwanami
    • 1
  • Kouji Uda
    • 1
  • Hiroshi Tada
    • 1
  • Tomohiko Suzuki
    • 1
  1. 1.Laboratory of Biochemistry, Faculty of ScienceKochi UniversityKochiJapan

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