title: Purification and characterization of beef pancreatic asparagine synthetase.
abstract: Bovine pancreatic asparagine synthetase has been partially purified using ammonium sulfate fractionation, DEAE ion-exchange, Cibacron Blue affinity chromatography, and HPLC anion-exchange chromatography to a specific activity of 170 nmol asparagine produced min-1 mg protein-1, or 1400-fold, from a crude homogenate. Using HPLC size exclusion chromatography, an apparent molecular weight of 110,000-120,000 was determined. An aspartyl-adenylate intermediate was found to occur by demonstrating an 18O transfer from [18O]Asp to AMP that was detected with 31P NMR. A number of divalent metals were found to be able to replace magnesium with retention of activity, but none produced as high an activity as Mg2+, and the stoichiometry of the ATP/Mg2+ ratio was found to be 1. The chloride ion was found to stimulate the glutamine-dependent and glutaminase reactions, but the ammonia-dependent reaction was inhibited. Chloride appeared to be a competitive inhibitor with respect to ammonia and produced negative cooperativity.
authors.forename: C A
authors.initials: CA
authors.lastname: Luehr
authors.forename: S M
authors.initials: SM
authors.lastname: Schuster
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation / Q000187:drug effects; D005972:Glutaminase / Q000378:metabolism; D008025:Ligases / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*
chemical_list: D002712:Chlorides; D008670:Metals; D005972:Glutaminase; D008025:Ligases; D001217:Aspartate-Ammonia Ligase