Separation, Purification, and Characterization of Two Isoforms of Glutamine Synthetase from Chlamydomonas reinhardii

Abstract

Two isoforms of glutamine synthetase. GS₁ and GS₂, have been separated from Chlamydomonas reinhardii cells grown autotrophically with nitrate. The intracellular level of GS₂ was higher than that of GS₁. In cells under darkness, the GS₁ peak increased markedly, whereas that of GS₂ became negligible. The two isoenzymes were purified to electrophoretic homogeneity by a method which included: a) DE-52 cellulose chromatography; b) ammonium sulphate fractionation: and c) affinity chromatography on ADP-sepharose. The specific activity was 114 and 63 U/mg for GS₁ and GS₂ respectively, and both enzymes (M_r = 380 000 and 373 000) are oligomeric proteins composed by 8 subunits of similar size (M_r = 48 000 in GS₁, and 46 000 in GS₂). The basic differences between GS₁ and GS₂ are: a) the effect of light on their intracellular level: b) their K_m for ammonium (83 and 244 μᴍ, respectively). Both isoenzymes were inhibited in a similar extent by ʟ-alanine, ʟ-glycine and ʟ-arginine.