Purification and characterization of glutamine synthetase from the unicellular cynabacterium Anacystis nidulans

Glutamine synthetase from the unicellular cynabacterium Anacystis nidulans was found associated with the membrane fraction of cell-free extracts. The enzyme could be solubilized by treatment of the cell membranes with the detergent alkyltrimethylammoniun and was purified to electrophoretical homogeneity by using affinity chromatography on 2′,5′-ADP-Sepharose. The molecular weight of the native enzyme was approx. 575000 but only a single protein band of 47 kDa was detected after sodium dodecyl sulphate gel electrophoresis, which implies a native enzyme complex with twelve identically sized subunits. Values for apparent Michaelis constant of the purified enzyme for ammonium, glutamate and ATP were 20, 5000 and 700 μM, respectively. Alanine behaved as an inhibitor of both activities (transferase and biosynthetic) of glutamine synthetase, whereas aspartate, leucine and lysine inhibited the biosynthetic activity of the enzyme, and glycine and serine only inhibited the transferase activity. Glutamate analogs, such as hydroxylysine, methionine sulfone, methionine sulfoximine and phosphinothricin, which inhibited ammonium uptake in vivo, behaved as potent inhibitors of glutamine synthetase in vitro. A. nidulans glutamine synthetase was inhibited by p-hydroxymercuribenzoate, the effect being reversed by treatment with dithioerythritol, dithiothreitol or mercaptoethanol.