Partial purification, characterization and properties of two isoforms of glutamine synthetase from Pennisetum glaucum L. leaves

Abstract

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 Co ²⁺.The pH optima for GS₁ and GS₂ were 7.0 and 8.0, respectively. GS₁ exhibited maximum activity at 42ºC, with activation energy of 18 KJ mol^-1 and a Q₁₀ of 3.0, whereas GS₂ showed maximum activity at 50ºC, with activation energy of 40 KJ mol^-1 and Q₁₀ of 2.25. GS₁ was more thermostable than GS₂. The K_m 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.