Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.
We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, alpha and beta, with molecular masses of approximately 40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKalpha and GKbeta were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKalpha and GKbeta chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKalpha, GKbeta, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKbeta enzyme, GKalpha was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKbeta, suggesting that GKalpha would have an activity similar to GKbeta if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.
Mizuta
Chisa
C
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan.
Tanaka
Kumiko
K
Suzuki
Tomohiko
T
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011077:Polychaeta / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C023839:glycocyamine phosphokinase; D003402:Creatine Kinase