Enzymic procedures for determining the average state of adenylylation of Escherichia coli glutamine synthetase

✍ Scribed by E.R. Stadtman; P.Z. Smyrniotis; J.N. Davis; Mary E. Wittenberger

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 875 KB
• Volume: 95
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90217-3

✦ Synopsis

Under physiological conditions, the activity of the glutamine synthetase in gram-negative bacteria is inversely proportional to the number of its subunits that are adenylylated [Kingdon, H. S., Shapiro, B. M., and Stadtman, E. R., (1%7), Proc. Nat. Acad. Sci. U. S. A. 58, 1703-17101. Six different enzymic procedures have been developed for determining the average state of adenylylation, i.e., the average number of adenylylated subunits per enzyme molecule, which can vary from 0 to 12. These methods depend on measurements of the yglutamyltransferase activity in assay mixtures containing Mn2+ at a pH where adenylylated and unadenylylated subunits are equally active and also under conditions where only unadenylylated subunits are active. The methods can be used to measure the state of adenylylation of glutamine synthetase in crude extracts with an accuracy of ?7%. MATERIALS Chemiculs L-glutamine, ADP, and AMP were obtained from Sigma Chemical Company; imidazole, 2-methylimidazole, 2,4-dimethylimidazole triethanolamine (TEA), and 3,3'dimethylglutaric acid (DMG) were from Aldrich Chemical Company; NH,OH * HCl, from Eastman; and KH2As0,, from Matheson-Coleman and Bell. The neutralized NH,OH reagent is prepared daily by mixing