Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi.
Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the obligate anaerobic archaebacterium Methanobacterium ivanovi. The 130-fold-purified enzyme was obtained by heat treatment, ion-exchange chromatography, and gel filtration. Like all other eubacterial GSs known so far, the GS of M. ivanovi was found to be a dodecamer of about 600,000 daltons composed of a single type of subunit. The enzyme was stable at 63 degrees C for 10 min and was not sensitive to oxygen. The isoelectric point was 4.6, and the optimum pH of gamma-glutamyltransferase activity was 8.0. The Km values for hydroxylamine, glutamine, and ADP in the transferase reaction were 6.8, 22.7, and 0.35 mM, respectively. L-Methionine-DL-sulfoximine strongly inhibited the activity. Like the GS from gram-positive bacteria, Anabaena sp., several yeasts, and mammals, the enzyme from M. ivanovi was not regulated by adenylylation as demonstrated by snake venom phosphodiesterase treatment. Inhibition of the transferase activity by L-alanine, glycine, L-histidine, and L-tryptophan was observed. L-Glutamine alone or in the presence of AMP did not inhibit the GS synthetic activity. The GS of Methanobacterium ivanovi did not cross-react with a variety of antisera against GS from Escherichia coli, Anabaena strain 7120, or Bacillus megaterium. Archaebacterial GS appears to be structurally and functionally similar to eubacterial GS in gram-positive bacteria.
Bhatnagar
L
L
Zeikus
J G
JG
Aubert
J P
JP
D002413:Cations, Divalent; D019605:Euryarchaeota / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000494:pharmacology; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
D002413:Cations, Divalent; D008717:Methionine Sulfoximine; D010727:Phosphoric Diester Hydrolases; D005974:Glutamate-Ammonia Ligase