Comparison of kinetic constants of creatine kinase isoforms.
The purpose of this study was to elucidate the functional differences between the CK isoforms by cloning the cDNAs of 12 CK isoforms: the M and B cytoplasmic forms and uMiCK from mouse, the M1, M2 and B cytoplasmic forms from Danio rerio, M1 and M2 cytoplasmic forms from the lower vertebrate Lampetra japonica, a cytoplasmic CK and a MiCK from the marine worm Neanthes diversicolor, and a cytoplasmic CK and a MiCK from the soft coral Dendronephthya gigantea. These were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and kinetic constants (K(m), K(d) and k(cat)) of all the recombinant enzymes, except for the unstable Dendronephthya cytoplasmic CK, were determined for the forward reaction. The kinetic constants of the M- and B-forms of the mouse and Danio cytoplasmic CKs differed significantly, with the K(m) for creatine (K(m)Cr) of M-CK being three- to nine-fold higher than that of B-CK, possibly reflecting differences in the concentration of creatine in muscle and brain cells. The mouse uMiCK had the lowest K(m)Cr value among the CK isoforms. In addition, it also exhibited a strong synergism for substrate binding (K(d)/K(m)=11.8). These results indicate that uMiCK has unique characteristics compared with other CK isoforms. Two subisoforms of M-CK were found in the lower vertebrate L. japonica, and the kinetic constants of recombinant M1- and M2-CKs differed significantly. The M1- and M2-CKs were expressed in skeletal muscle with a ratio of 7:3, while M1-CK was the predominant subisoform in the testis. The kinetic constants of cytoplasmic CK from the marine worm Neanthes were significantly different from those of Neanthes MiCK, possibly indicating that functional differences among CK isoforms occurred at least before the divergence of annelids from other protostome invertebrates.
Matsushima
Kazuki
K
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan.
Uda
Kouji
K
Ishida
Keiko
K
Kokufuta
Chinatsu
C
Iwasaki
Nozomu
N
Suzuki
Tomohiko
T
D000818:Animals; D037421:Anthozoa / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D002384:Catalysis; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D052276:Creatine Kinase, BB Form / Q000235:genetics / Q000378:metabolism*; D052283:Creatine Kinase, MM Form / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D007700:Kinetics; D007798:Lampreys / Q000378:metabolism; D008297:Male; D058619:Maltose-Binding Proteins; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D009928:Organ Specificity; D011077:Polychaeta / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013737:Testis / Q000201:enzymology; D015027:Zebrafish / Q000378:metabolism
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D003402:Creatine Kinase; D052276:Creatine Kinase, BB Form; D052283:Creatine Kinase, MM Form