Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.

Abstract

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, α and β, with molecular masses of ∼40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKα and GKβ 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 GKα and GKβ chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKα, GKβ, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKβ enzyme, GKα 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 GKβ, suggesting that GKα would have an activity similar to GKβ 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.

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.