title: Purification and properties of glutamine synthetase from Nocardia asteroides.
abstract: Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn2+ and Mg2+ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50 degrees C. Amino acids such as L-alanine, glycine, and aspartate inhibited the GS activity. The Km values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 microM, 7.7 mM, and 200 microM, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.
authors.affiliation: Department of Biology, Fisk University, Nashville, TN 37208, USA.
authors.forename: C
authors.initials: C
authors.lastname: Palaniappan
authors.forename: M
authors.initials: M
authors.lastname: Gunasekaran
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium; D008345:Manganese; D008970:Molecular Weight; D009616:Nocardia asteroides / Q000201:enzymology*; D013696:Temperature
chemical_list: D000596:Amino Acids; D000643:Ammonium Chloride; D008345:Manganese; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium