The overall goal of this project is an elucidation of the possible molecular events that led to the mitchondrial compartmentation of glutamine synthetase in liver tissue during vertebrate evolution. This localization of the enzyme allowed for the development of uricoteley as a major kind of excretory nitrogen metabolism. Most mitochondrial proteins are coded for in the nucleus, translated in cytosol, and targeted to mitochondria by N-terminal signal sequences. The larger size of the pre-enzyme allows it to be distinguished from the mature enzyme. We have shown this to probably be true for elasmobranch liver mitochondrial glutamine synthetase and we now intend to characterize the import of the pre-enzyme by elasmobranch liver mitochondria in vivo and in vitro. We have also found that in birds and alligators there is no detectable size difference between the nascent and mature glutamine synthetase peptide in liver, despite its mitochondrial matrix localization in tissue. In order to determine if there is an internal targeting sequence in the avian enzyme, we will clone and sequence glutamine synthetase mRNA cDNA from both liver and brain. The enzyme remains in the cytosol in the latter tissue so a comparison of the deduced amino acid sequences for the two should give some clue as to the nature and location of the signal sequence used for import of the liver enzyme. We also will obtain the deduced sequences for elasmobranch and possibly tortoise pre-glutamine synthetase for comparative purposes. This comparison should give some indication of the possible changes that have occured during evolution that led from a classical presequence mechanism for targeting of the enzyme to liver mitochondria in primitive vertebrates to whatever the mechanism used in higher uricotelic vertebrates. This research on the molecular mechanisms of how enzymes are targeted to specific cell compartments is significant to cell biology and biochemistry. The regulation of enzyme activity is a key question for understanding metabolism.//
