Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.