Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.
Introduction
Members of the phosphagen kinase enzyme family play a key role in the regulation of energy production and utilization in animals (Morrison, 1973, Wyss et al., 1992, Wyss and Kaddurah-Daouk, 2000, Ellington, 2001). These enzymes catalyze the reversible transfer of high-energy phosphoryl groups from ATP to naturally occurring guanidine compounds, producing phosphorylated high-energy guanidine compounds referred to as phosphagens.
While phosphocreatine (PCr)/creatine kinase (CK) is the only phosphagen/phosphagen kinase pair found in vertebrates, various phosphagens and corresponding enzymes are found in invertebrates (Morrison, 1973), including PCr/CK; phosphoglycocyamine (PGlyc)/glycocyamine kinase (GK); phosphotaurocyamine (PTau)/taurocyamine kinase (TK); phosphohypotaurocyamine (PHypo)/hypotaurocyamine kinase (HTK); phospholombricine (PLom)/lombricine kinase (LK); phosphoopheline (POph)/opheline kinase (OK); phosphothalassemine (PTha)/thalassemine kinase (ThaK); and phosphoarginine (PArg)/arginine kinase (AK).
Although the evolutionary processes are not fully understood, CK, GK, TK, and LK appear to have evolved from a common ancestor (Muhlebach et al., 1994, Suzuki and Furukohri, 1994, Suzuki et al., 1997), and the cytoplasmic forms of these four enzymes are known to have a conventional dimeric structure consisting of two 40-kDa subunits. The structure and function of CK have been well characterized and the presence of at least three isoforms (cytoplasmic, flagellar, and mitochondrial) has been confirmed in the ancestral CK gene (Suzuki et al., 2004). The functional properties of GK, TK, LK, HTK, OK, and ThaK are not well known, and, interestingly, these enzymes are found only in annelid-like worms.
On the other hand, AK is the most widely distributed in invertebrates; AK activity has been identified in protozoa (Noguchi et al., 2001) and its gene has been found in the genomes of Paramecium sp. and Tetrahymena sp. AK may occur, in different species, either as a monomeric or a dimeric enzyme (Morrison, 1973). The amino acid sequence and gene structure (exon/intron organization) of AK are rather distant from those of CK and CK-related enzymes, suggesting that AK and CK diverged at an early stage of evolution (Muhlebach et al., 1994, Suzuki and Furukohri, 1994, Suzuki et al., 2004).
Occurrence of more than one phosphagen (and its corresponding phosphagen kinase) in an animal was first described by Needham et al. (1932), and three decades later, Robin (1964) defined this as “pluriphosphagen.” The occurrence of more than one phosphagen in an animal is widely observed in polychaetes (e.g., PArg and PTau in Amphitrite sp.; PGlyc and PCr in Nereis sp.; PLom and PArg in Notomastu sp.; and PTau, PArg, and PCr in Mercierella sp.) and in selected species of echinoderms and tunicates (Ratto et al., 1989). However, it is not clear whether the different phosphagens and corresponding kinases are localized in the same cell.
The first detailed research on GK was done by Pradel et al. (1968) who observed that GK from the polychaete Nephthys coeca is a dimer of two nearly identical subunits with native masses of 89 kDa. We showed that GK isolated from the body wall musculature of the polychaete Neanthes diversicolor is a heterodimer consisting of two subunits, denoted α and β, that differ slightly in molecular mass (Furukohri and Suzuki, 1987). Furthermore, we amplified cDNAs coding for these two subunits: the α chain codes for a 375-residue protein, whereas the β chain codes for a 391-residue protein (Suzuki et al., 1999). In addition to a 16-residue extension on the β subunit, there are several amino acid substitutions in the ∼50 residue N-terminal region. The amino acid sequences for the remaining regions are identical (Suzuki et al., 1999, Ellington et al., 2004). Very recently, we showed that the α and β chains of N. diversicolor GK are generated by alternative splicing (Ellington et al., 2004).
In the present study, we isolated native GK and CK enzymes from the tropical marine worm Namalycastis sp. and determined the sequences of the GK α and β chain cDNAs. We also cloned them into the pMAL plasmid, which allowed its expression in Escherichia coli as maltose-binding protein (MBP) fusion protein. We then purified the MBP-GK fusion proteins and determined their kinetic parameters. Based on these studies, we propose a physiological role of the three enzymes, GK, cytoplasmic CK, and mitochondrial CK (MiCK) in Namalycastis, in the pluriphosphagen kinase system.
Section snippets
Isolation of GK and CK from Namalycastis sp.
Namalycastis sp. was purchased from the Tateno fishery shop of Yokohama, Japan. All procedures were performed at 4 °C as far as possible. Whole animals (total 35 g for two specimens) of the marine worm Namalycastis sp. were homogenized with 175 ml of 10 mM Tris–acetate buffer, pH 8.1, containing 0.1 mM dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF). The tissue extract was fractionated with 50–80% saturated ammonium sulfate. The precipitate was dissolved in a minimum volume of 10
Isolation of Namalycastis GK and CK
The crude aqueous extracts of Namalycastis sp. were passed through a Sephacryl S-200 gel filtration column, and the fraction, possessing GK and CK activities, was pooled and applied to a DEAE-5PW ion exchange column (Fig. 1). Four peaks were clearly isolated on the DEAE column, and peak 1 contained GK activity. Reducing SDS-PAGE showed that the isolated GK was highly purified and that it consists of equimolar proportions of two distinct subunits, α and β, with molecular masses of ∼40 kDa (Fig. 1
Acknowledgements
We thank Drs. T. Miura and T. Furukohri for the identification of Namalycastis. We also thank anonymous referees for giving valuable comments and suggestions.
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