PURIFICATION AND PROPERTIES OF AZOTOBACTER-VINELANDII GLUTAMINE-SYNTHETASE

A procedure is described for the purification of glutamine synthetase from the nitrogen-fixing organism Azotobacter vinelandii. Electron micrographs of the enzyme reveal a dodecameric arrangement of its subunits in two superimposed hexagonal rings similar to the glutamine synthetase of Escherichia coli. Disc eleetrophoresis in the presence of sodium dodecyl sulfate and sedimentation studies show a subunit molecular weight of 56,500 and a sedimentation coefficient (s20,w) of the native enzyme of 20.0 S. Like the E. coli enzyme, the glutamine synthetase of A. vinelandii is regulated by adenylylation/deadenylylation. This finding was derived from (a) studies on the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of enzyme isolated from cells grown on a nitrogen-rich medium, (b) the identification of the AMP released by the phosphodiesterase by thin-layer chromatography, (c) the selective precipitation of adenylylated enzyme with antibodies directed against adenylylated bovine serum albumin, and (d) the in vitro incorporation of radioactivity from [14C]ATP into deadenylylated enzyme in the presence of either crude extract from A. vinelandii or partially purified adenylyl transferase from E. coli. The state of adenylylation appears to have a similar influence on the catalytic properties of A. vinelandii glutamine synthetase as on those of the E. coli enzyme, with the exception that the deadenylylated form of the A. vinelandii glutamine synthetase is almost inactive in the Mn-dependent transferase reaction. © 1979.