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The chemistry of human and bovine asparagine synthetase
Abstract
The optimal growth and immunoaffinity purification conditions were determined for the isolation of recombinant human asparagine synthetase from Saccharomyces cerevisiae. An analysis of the glutamine and aspartyl-AMP binding sites of the human protein was completed with the aid of bovine-specific monoclonal antibodies and the L-glutamine and ATP substrate analogs, 6-diazo-5-oxo-L-norleucine and 8-azidoadenosine-5$\sp\prime$-triphosphate, respectively. The modification of the glutamine binding site with DON resulted in a loss of the glutamine-dependent and glutaminase activities of the enzyme, while the ammonia-dependent activity was unaffected. The modification of the ATP binding site with 8-N$\sb3$ATP inhibited the glutamine-dependent activity, leaving the glutaminase activity unaltered. The binding domains recognized by the monoclonal antibodies were investigated, with respect to the native and chemically modified enzyme, through enzymatic activity assays and ELISA procedures. The experiments demonstrated the existence of two discrete catalytic domains within the human protein. The epitope region of human asparagine synthetase was observed to vary significantly from that of either the bovine or rodent enzymes. The nucleotide specificity and anion/cation dependence of the wild-type human asparagine protein was examined and the kinetic parameters were determined for the gene products of the pVTXAS1 and pGAT21 recombinant constructs. The ATP hydrolysis and inorganic pyrophosphate exchange reactions catalyzed by bovine pancreatic asparagine synthetase were studied and discussed in terms of possible mechanistic pathways. Asparagine synthetase was investigated from normal and lymphatic murine thymic tissue. The enzymatic activity and structural integrity of the protein was examined by HPLC and Western blot analyses. The relationship between dietary levels of L-asparagine and the catalytic activity of rodent asparagine synthetase was studied. The asparagine synthetase enzymatic activity was examined from hepatic tissue extracts of the mice and rats, prior to and following 52 days on diets either fortified or deficient in L-asparagine.
Subject Area
Biochemistry
Recommended Citation
Larsen, Michele Campaigne, "The chemistry of human and bovine asparagine synthetase" (1991). ETD collection for University of Nebraska-Lincoln. AAI9133296.
https://digitalcommons.unl.edu/dissertations/AAI9133296