Characterization and sequencing of glutamine synthetase cDNA from liver ofthe ureotelic gulf toadfish (Opsanus beta)

The hepatic enzyme, glutamine synthetase (GSase) is a pivotal protein in th e regulation of urea synthesis in fish. The sequence of the DNA encoding fo r GSase from liver of the ureotelic gulf toadfish (Opsanus beta) was analyz ed through a suite of molecular techniques (including cDNA cloning, RACE PC R, and genomic PCR). An open reading frame (ORF) was identified in the cDNA sequence which codes for a protein of 394 amino acids with high identity ( 86%) to dogfish shark GSase. In the course of generating a suitable probe, a partial sequence was also obtained for horned shark GSase which also had high identity with the dogfish shark gene (93%). Like the dogfish shark GSa se, the toadfish gene has two methionine translation initiation sites; the downstream site apparently codes for a cytoplasmic isozyme, while the upstr eam site adds an N-terminal peptide leader sequence of 23 amino acids to th e 'cytoplasmic' protein. This leader sequence has characteristics consisten t with a mitochondrial targeting peptide, including a cleavage recognition motif (Arg-X-Phe) and the apparent ability to form an amphiphathic helix. N orthern analysis revealed that there is a single predominant transcript of similar to 2 kb in size. These results are consistent with the interpretati on that in the gulf toadfish GSase cytoplasmic and mitochondrial isozymes a re coded for by a single gene and mRNA transcript which is differentially t ranslated at either initiation site. These results are discussed in the con text of prior results for enzyme kinetic characteristics and urea synthesis /excretion physiology. (C) 1999 Elsevier Science Inc. All rights reserved.