Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
ASPARAGINE METABOLISM IN EUCARYOTIC CELLS (GENETICS, OMEGA AMIDASE, SYNTHETASE)
Abstract
Neurospora crassa mutants deficient in asparagine synthetase were selected using the procedure of inositol-less death. Over 100 mutants were isolated whose singular difference from wild-type appeared to be a specific requirement for asparagine (asn). Among these, 11 were found to be temperature-conditional. In vitro assays with heterokaryons indicated that the mutation is a dominant one. Enzyme assays of temperature stabilities of cell-free extracts from temperature-sensitive strains indicated that the mutations were in the structural gene(s) for asparagine synthetase. Incubation of the wild-type enzyme with ATP prior to assay caused a two-fold activation in specific activity of the asparagine synthetase. Further work involving asparagine catabolism in the isolated rat liver mitochondria showed asparagine breakdown via transamination to (alpha)-ketosuccinamide ((alpha)KSA). Using asparagine and glyoxylate as substrates for a mitochondrial asparagine-glyoxylate transaminase yields (alpha)-ketosuccinamide and glycine. The (alpha)-ketosuccinamide is deaminated by (omega)-amidase to yield OAA. Because OAA inhibits succinate dehydrogenase, its production can be followed by monitoring decreased oxygen uptake during oxidation in mitochondria. The addition of (alpha)KSA to isolated mitochondria oxidizing succinate yields an even greater reduction of oxygen consumption. HPLC separation of (alpha) keto acids and amino acids allowed direct observation of label flow using ('14)C-asparagine. The production of labeled (alpha)KSA, OAA and CO(,2) from ('14)C-asn was monitored and the results were consistent with the proposed mitochondrial asn catabolism pathway. Assays of cell-free mitochondrial extracts showed no detectable asparaginase activity. Studies with cytosolic (omega)-amidase from rat liver indicated that a major pathway for asn breakdown in the cytosol could include (alpha)KSA hydrolysis to OAA and ammonia. Cytosolic and mitochondrial (omega)-amidases from rat liver were partially purified. The two enzymes were found to have distinctly different characteristics. The cytosolic (omega)-amidase has a pH optimum of 9 while the mitochondrial enzyme optimum is pH 8. With (alpha)KSA, the K(,m) for the cytosolic enzyme ws 2.6 mM in the presence of MgCl(,2) while the mitochondrial enzyme exhibited negative cooperativity. With (alpha)-ketoglutaramide ((alpha)KGA) as the substrate, the cytosolic enzyme showed negative cooperativity in the presence of both MgCl(,2) and CaSO(,4). The K(,m) for the mitochondrial enzyme with (alpha)KGA was 6.8 mM. With (alpha)KSA in the presence of CaSO(,4), however, the cytosolic enzyme demonstrated positive cooperativity. The cytosolic enzyme is stabilized by Mg('2+) or Ca('2+) but the membrane-bound mitochondrial enzyme utilizes KH(,2)PO(,4). Both enzymes are inhibited by sulfhydryl reagents.
Subject Area
Biochemistry
Recommended Citation
MACPHEE, KATHLEEN G, "ASPARAGINE METABOLISM IN EUCARYOTIC CELLS (GENETICS, OMEGA AMIDASE, SYNTHETASE)" (1984). ETD collection for University of Nebraska-Lincoln. AAI8412311.
https://digitalcommons.unl.edu/dissertations/AAI8412311