Cytoplasmic and Mitochondrial Creatine Kinases from the Skeletal Muscle of Sperm Whale (Physeter macrocephalus). Molecular Cloning and Enzyme Characterization
- 102 Downloads
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.
KeywordsCreatine kinase Mitochondrial Phosphagen kinase Guanidino kinase Kinetic constant Cetacean Sperm whale Physeter macrocephalus
muscle-type isoform of CK
brain-type isoform of CK
sarcomeric mitochondrial isoform of CK
ubiquitous mitochondrial isoform of CK
creatine; MBP, maltose-binding protein
This work was supported by a Grant-in-Aid for Scientific Research in Japan to TS (17570062).
- 4.Schlattner U, Tokarska-Schlattner M, Wallimann T (2005) Biochim Biophys Acta 1762:164–180 Google Scholar
- 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
- 9.Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM (1992) Biochem J 281:21–40Google Scholar
- 11.Dolar ML, Suarez P, Ponganis PJ, Kooyman GL (1999) J Exp Biol 202:227–236 Google Scholar
- 13.Borson ND, Salo WL, Drewes LR (1992) PCR Methods Appl 2:144–148Google Scholar
- 18.Morrison JF, James E (1965) Biochem J 97:37–52Google Scholar