Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum.
Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.
Singhal
R K
RK
Biochemistry Laboratory, Indian Institute of Technology, New Delhi.
Krishnan
I S
IS
Dua
R D
RD
D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003013:Clostridium / Q000201:enzymology*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005970:Glutamate Synthase / Q000032:analysis* / Q000302:isolation & purification; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity; D000637:Transaminases / Q000032:analysis*
D010446:Peptide Fragments; D007501:Iron; D005970:Glutamate Synthase; D000637:Transaminases