Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp
| Autor: | Tomohiko Suzuki, Chisa Mizuta, Kumiko Tanaka |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
DNA
Complementary Physiology Recombinant Fusion Proteins Molecular Sequence Data Biology Biochemistry chemistry.chemical_compound Complementary DNA Escherichia coli Animals Enzyme kinetics Amino Acid Sequence Cloning Molecular Molecular Biology Polyacrylamide gel electrophoresis Peptide sequence Creatine Kinase chemistry.chemical_classification Phosphotransferases (Nitrogenous Group Acceptor) Polychaeta Arginine kinase Molecular biology Amino acid Glycocyamine chemistry biology.protein Creatine kinase |
| Zdroj: | Comparative biochemistry and physiology. Part B, Biochemistrymolecular biology. 140(3) |
| ISSN: | 1096-4959 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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