ELLINGTON, W.R.; Florida State Univ.: Cloning and Expression of a Polychaete Glycocyamine Kinase

Glycocyamine (guanidinoacetate) is the direct precursor of creatine, the guanidine substrate for the creatine kinase (CK) reaction. In many species of errant polychaetes (Nereidae, Nephthydidae, Aphroditidae) glycocyamine is present at high tissue levels. However, this compound is not methylated to form creatine but rather is phosphorylated to the phosphagen glycocyamine phosphate (GP) by glycocyamine kinase (GK). This enzyme is a member of the highly conserved phosphagen kinase family which includes CK and arginine kinase. GK activities are quite high in the body wall of the sand worm Nereis virens. We have obtained the full length cDNA sequence for Nereis GK by RTPCR amplification of body wall total RNA using a “universal” phosphagen kinase primer and 3′-end sequence derived gene specific primers. The cDNA consists of 1759 bp with an open reading frame of 1,122 bp and 5′ and 3′ untranslated regions of 19 bp and 618 bp, respectively (sequence deposited in GenBank accession no. AY05911182). The cDNA codes for a 374 residue protein with a calculated Mr of 42.8 kD and theoretical pI of 6.61. A cDNA for the open reading frame, with start and stop codons, was obtained by PCR, ligated into the pETBlue1 expression vector and transfected into NovaBlue cells. Plasmids containing the insert in the correct orientation and free of PCR-induced errors were used to transform DE3 cells. Expression of protein yielded soluble GK activity. Recombinant protein will ultimately be utilized for developing ultra-sensitive assays of tissue/cellular levels of glycocyamine and activities of arginine:glycine amidinotransferase in on-going studies of creatine-phosphocreatine homeostasis in invertebrate and lower chordate systems. Supported by NSF grant IBN-0130024.