Methods for purifying to homogeneity glutamine synthetases (l-glutamate:ammonia ligase (ADP-forming), EC 18.104.22.168) of pea seed cytosol (GScs) and leaf cytosol (GScl) have been developed. The gel filtration method was used to estimate GScs molecular weight as 384 000 and that of GScl, 520 000. Under electrophoresis in polyacrylamide gel in the presence of SDS, GScs and GScl dissociated into monomers of molecular weight 48 000 and 64 000, respectively. The quaternary structures of GScs and GScl were investigated by electron microscopy. The two enzymes were found to consist of eight identical monomers each arranged with dihedral point group symmetry 422 (D4) at the vertices of two squares. These squares are twisted about the 4-fold axis relative to each other. Unlike GScl; GScs is characterized by annular projection of particles in micrographs due to filling of the interior portion of the protein molecule with a stain. GScs and GScl secondary structures, calculated on the basis of the circular dichoroism spectra, also differed. The nature of the effects of substrates, M2+, and methionine sulphoximine on GScs secondary structure was investigated. The isoelectric point of GScs was situated at pH 5.15, and that of GScl at pH 4.8. The N-terminal amino acid of GScl was alanine, and that of GScs, glycine. The kinetic characteristics of GScl and GScs also differ. GScs and GScl, and also leaf chloroplast glutamine synthetase and root glutamine synthetase of pea plant were compared by means of rabbit antibodies against GScs. The conclusion was made that the multiple molecular forms of glutamine synthetase of pea plant may be isoenzymes.
|Original language||English (US)|
|Number of pages||15|
|Journal||Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular|
|State||Published - Apr 29 1985|
- (Pea leaf, seed)
- Electron microscopy
- Glutamine synthetase