Isolation and properties of isocitrate lyase from Lupinus seed

Isocitrate lyase (threo-d_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) was purified from cotyledons of Lupinus seedlings. The final preparation showed two bands after polyacrylamide-gel electrophoresis. The optimum pH using phosphate, Tris or imidazole buffer was at pH 7.5; with triethanolamine (TRA) it was at pH 7. The enzyme required Mg²⁺ for maximal activity, and N-ethylmaleimide (NEM) inactivated the enzyme. Activity was increased by incubation with the reducing agents, glutathione (GSH), acetylcysteine (acetylcys), dithionite (Na₂S₂O₄), thioglycolate (TG) or 1,4-dithioerythritol (DTE). Na₂S₂O₄ and DTE were the most active among the tested substances and DTE prevented much of the inactivation by NEM. The apparent K_m value for isocitrate was ca 1 mM in phosphate buffer at pH 6.8 or 7.5 but was substantially lower (0.1–0.2 mM) using Tris, TRA or imidazole buffers. Glyoxylate, oxalate and malonate were competitive inhibitors of the enzyme. Synthase activity of the enzyme (i.e. formation of isocitrate from succinate and glyoxylate) was demonstrated. The K_m values for glyoxylate and succinate were 0.05 and 0.2 mM, respectively. The addition of glyoxylate to the culture medium in which Lupinus seeds germinate resulted in a reduced development of isocitrate lyase activity during germination.