Partial purification, characterization and properties of two isoforms of glutamine synthetase from Pennisetum glaucum L. leaves.
Two isozymes of glutamine synthetase GS1 and GS2 were partially purified from Pennisetum glaucum leaves by ion-exchange and gel filtration chromatography and their kinetic and regulatory properties were studied using semisynthetase assay of GS. Mg2+ was the most effective cation for activity of both the isozymes; however, it could be efficiently replaced by Co2+. The pH optima for GS1 and GS2 were 7.0 and 8.0, respectively. GS1 exhibited maximum activity at 42 degrees C, with activation energy of 18 KJ mol(-1) and a Q10 of 3.0, whereas GS2 showed maximum activity at 50 degrees C, with activation energy of 40 KJ mol(-1) and Q10 of 2.25. GS1 was more thermostable than GS2. The Km value for Mg2+ of GS1 was 2-fold higher than GS2; however, these isozymes did not differ much in their affinity for other substrates. Alanine, serine and glycine lowered GS1 and GS2 activities, whereas cysteine enhanced their activities with a more pronounced effect on GS2. Serine inhibited the activity of both the isoforms in a competitive-manner, whereas alanine was a non-competitive inhibitor, with respect to glutamate. AMP and ADP were competitive inhibitor with respect to ATP for both the isozymes.
College of Basic Sciences, CCS Haryana Agricultural University, Hisar 125 004, India. ghoshshilpi@hotmail.com
Shilpi
S
Ghosh
D000244:Adenosine Diphosphate / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D002736:Chloroplasts / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D031782:Pennisetum / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology*; D018517:Plant Roots / Q000378:metabolism; D020033:Protein Isoforms; D012639:Seeds / Q000378:metabolism; D013696:Temperature
D007527:Isoenzymes; D020033:Protein Isoforms; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase