Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle.
In vertebrates, the creatine kinase isoenzyme family consists of four types of isoforms: cytosolic muscle type (M-CK), cytosolic brain type (B-CK), mitochondrial ubiquitous, acidic type (Miu-CK), and mitochondrial sarcomeric, basic type (Mis-CK). Until recently, the existence of more than one subisoform of CK isoenzyme has been demonstrated only in fishes by starch gel electrophoresis. We report herein the isolation of three full-length cDNAs that correspond to three closely related creatine kinase M-CK genes from common carp (Cyprinus carpio), designated the M1-CK, M2-CK, and M3-CK genes. Using oligonucleotide probes that correspond to the same region but with the most variable sequences, different restricted genomic hybridization patterns have been obtained. These Southern blot results indicate that the three cDNAs come from different genes. Northern blot analysis using probes that correspond to the 3'-untranslated regions further show that all three subisoforms are expressed specifically in carp muscle. The deduced amino acid sequences of these three subisoforms of carp M-CK show about 85% identity to mammalian M-CK isoenzyme. Finally, the three cDNAs have been expressed in Escherichia coli with a molecular mass of approximately 43,000 Da, and these recombinant proteins exhibit creatine kinase activity. All of these data suggest that the M-CK isoenzymes have at least three subisoforms in carp.
Sun
H W
HW
Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China.
Hui
C F
CF
Wu
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D003402:Creatine Kinase