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ISOLATION AND CHARACTERIZATION OF ASPARAGINE/GLYOXYLATE AMINOTRANSFERASE
Abstract
Experimental observations previously published suggest that asparagine and glycine may be metabolically linked. In order to probe this possible link, a series of experiments were performed. Asparagine synthetase activity was found to be stimulated by methotrexate (MTX). MTX was then postulated to act indirectly to eliminate glycine synthesis from serine and cause asparagine-dependent glycine synthesis to become more significant for cell survival. Thus, there was an increased demand for asparagine which resulted in an increased level of asparagine biosynthesis. To probe further the metabolic link between asparagine and glycine metabolism, the possibility was explored that asparagine could provide nitrogen for glycine synthesis via an aminotransferase reaction. Transamination between asparagine and glyoxylate yields (alpha)-ketosuccinamide and glycine. The liver asparagine/glyoxylate aminotransferase was found to have the highest enzymatic activity. Therefore, the hepatic isozymes of the aminotransferase were isolated and characterized. The cytosolic and mitochondrial isozymes have significantly different heat stabilities, amino acid and keto acid specificities. Since the mitochondrial isozyme predominated in hepatic tissue and was more readily purified, it was studied kinetically. The mitochondrial isozyme was found to be unusual in its kinetic behavior. While the majority of isolated transaminases exhibit ping pong mechanisms, this enzyme showed an ordered bi bi kinetic pattern in steady state measurements. Upon completion of pre-steady state kinetic measurements, the mechanism was found to be more complex than the simple ordered mechanism. With this enzyme, marked substrate inhibition was observed with asparagine which decreased with increasing pyruvate. The enzyme also showed strong negative cooperativity with pyruvate. A significant lag was observed in the pre-steady state experiments. The lag showed very complicated dependencies on substrates. A model for the asparagine aminotransferase was proposed which requires two enzyme forms to explain all the kinetic data presented. Data are also presented suggesting that (alpha)-ketoglutarate can be hydroxylated and cleaved to provide the carbon skeleton for asparagine-dependent glycine synthesis.
Subject Area
Biochemistry
Recommended Citation
MAUL, DIANA MARIE, "ISOLATION AND CHARACTERIZATION OF ASPARAGINE/GLYOXYLATE AMINOTRANSFERASE" (1983). ETD collection for University of Nebraska-Lincoln. AAI8318667.
https://digitalcommons.unl.edu/dissertations/AAI8318667