ASPECTS OF AMMONIAGENESIS IN THE CHANNEL CATFISH ICTALURUS PUNCTATUS
CASEY, CAROL ANN
Doctor of Philosophy
An investigation of the cellular mechanism for ammoniagenesis in a freshwater teleost, the channel catfish, provided the basis for research performed in this thesis. Activities and subcellular localizations of enzymes associated with nitrogen metabolism were measured in catfish liver. Glutamate dehydrogenase (the enzyme responsible for ammonia generation during transdeamination) and AMP deaminase (the ammonia releasing enzyme of the purine nucleotide cycle) were both present in quantities sufficient to account for ammoniagenesis by the catfish. Neither glutamine synthetase nor carbamyl phosphate synthetase (enzymes responsible for "detoxication" of intramitochondrially generated ammonia in uricoteles and ureoteles) were detectable in catfish liver mitochondria. The role of the purine nucleotide cycle was investigated through the use of heavy isotope tracer studies. If the purine nucleotide cycle were operative in fish liver, incubation of hepatocytes with ('15)N alanine should result in the incorporation of the amino nitrogen of alanine into the 6-amino function of AMP (an intermediate in the purine nucleotide cycle). No significant incorporation of the heavy isotope was observed in either the total adenine nucleotide pool or the smaller AMP pool. Experiments using hepatocytes from both the rat (a ureotele) and the chicken (a uricotele) likewise did not show incorporation. End-products isolated from hepatocyte preparations of all three animals, however, exhibited marked incorporation (over 80 percent) of the heavy isotope. Based on these results, a major contribution by the purine hucleotide cycle to ammoniagenesis in fish was ruled out. Preliminary studies utilizing isolated mitochondria examined the alternative pathway for ammoniagenesis, transdeamination. Isolated catfish mitochondria liberated ammonia from glutamate, glutamine, and alanine, although the mitochondrial rates were lower than those obtained using hepatocytes. Various metabolic inhibitors (bromofuroate, an inhibitor of glutamate dehydrogenase; and aminoxyacetate, which inhibits transminases) were used to determine their effect on the capability of mitochondria to produce ammonia. The results obtained in these studies imply a central role for glutamate dehydrogenase during both oxidation and ammonia release from amino acids. Data presented in this thesis effectively eliminate the purine nucleotide cycle as a viable mechanism for catabolizing amino acids in catfish liver. If transdeamination is the pathway utilized in fish during ammoniagenesis, the process of ammonia release from the mitochondria may be different than the mechanism used by ureoteles or uricoteles.