Elasmobranch glutamine synthetase: Synthesis and subcellular localization of tissue-specific isozymes
Ritter, Nadine Marie
Doctor of Philosophy
Elasmobranchs (sharks, skates and ray) synthesize and retain high amounts of urea for the purpose of osmoregulation. Ureosmotics utilize a pathway for the production of urea that incorporates enzymes used in the formation of urea and uric acid in higher vertebrates. Ammonia is produced, and the first steps in its detoxification and processing for excretion occur, in the mitochondria of the liver of these animals, as it does in ureoteles and uricoteles. The enzymes involved in these steps are therefore required to be localized in the mitochondria. Most mitochondrial enzymes are encoded by nuclear genes, synthesized in the cytoplasm, and imported into the organelle. In some instances, mitochondrial enzymes have a cytosolic counterpart. The majority of compartmental isozymes are generally not related at either the structural or genetic level. The objectives of this work were to investigate the tissue-expression and localization of glutamine synthetase, the enzyme that detoxifies intramitochondrially-generated ammonia in elasmobranchs. It was determined that glutamine synthetase was mitochondrial in the liver, but cytosolic in the brain of elasmobranchs and a holocephalan. These compartmental isozymes were shown to have different subunit sizes. This work described the first report of glutamine synthetase isozymes in vertebrates. The two compartmental isozymes have the same isoelectric points and antibody specificity, suggesting a similar molecular derivation. Mitochondrial glutamine synthetase was shown to be translated with a leader sequence that is removed during import, while the cytosolic form is translated as the mature size. In the stingray and shark, it was found that a single dominant RNA species gives rise to the tissue-specific isozymes in each animal. Preliminary work suggests the isozymes are encoded by a single gene.