Purification and characterization of beef pancreatic asparagine synthetase |
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| Authors: | C A Luehr S M Schuster |
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| Institution: | 1. Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan;2. Department of Pharmacology, Fukushima Medical University, Fukushima, Japan;3. First Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan;1. Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy;2. IRCCS, Centro Cardiologico Monzino, Milan, Italy;3. IRCCS, Humanitas Research Foundation, Bruzzano, Milan, Italy;4. IRCCS Multimedica, Milan, Italy;5. SISA Centre for the Study of Atherosclerosis, Bassini Hospital, Cinisello B, Milan, Italy;6. William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University, London, UK |
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| 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. |
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