The chemistry of human and bovine asparagine synthetase

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\sb3ATP 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

PAHs Target Hematopoietic Linages in Bone Marrow through Cyp1b1 Primarily in Mesenchymal Stromal Cells but Not AhR: A Reconstituted In Vitro Model

7,12-Dimethylbenz(a)anthracene (DMBA) rapidly suppresses hematopoietic progenitors, measured as colony forming units (CFU), in mouse bone marrow (BM) leading to mature cell losses as replenishment fails. These losses are mediated by Cyp1b1, independent of the AhR, despite induction of Cyp1b1. BM mesenchymal progenitor cells (MPC) may mediate these responses since basal Cyp1b1 is minimally induced. PreB colony forming unit activity (PreB CFU) is lost within 24 hours in isolated BM cells (BMC) unless cocultured with cells derived from primary MPC (BMS2 line). The mouse embryonic OP9 line, which provides more efficient coculture support, shares similar induction-resistant Cyp1b1 characteristics. This OP9 support is suppressed by DMBA, which is then prevented by Cyp1b1 inhibitors. OP9-enriched medium partially sustains CFU activities but loses DMBA-mediated suppression, consistent with mediation by OP9 Cyp1b1. PreB CFU activity in BMC from Cyp1b1-ko mice has enhanced sensitivity to DMBA. BMC gene expression profiles identified cytokines and developmental factors that are substantially changed in Cyp1b1-ko mice. DMBA had few effects in WT mice but systematically modified many clustered responses in Cyp1b1-ko mice. Typical BMC AhR-responsive genes were insensitive to Cyp1b1 deletion. TCDD replicated Cyp1b1 interventions, suggesting alternative AhR mediation. Cyp1b1 also diminishes oxidative stress, a key cause of stem cell instability