Characterization of isocitrate lyase from the yeast Yarrowia lipolytica

The active form of isocitrate lyase (ICL) from the yeast Yarrowia lipolytica was eluted as single peak from ion exchange on DEAE-cellulose. The enzyme had a specific activity of 7.4 U/mg. Its molecular mass was estimated to be approximately 200 to 210 kDa by gel filtration chomatography on Sephadex G 200. In SDS-polyacrylamide gel electrophoresis the enzyme was characterized by an unique protein band of about 50 kDa, thus indicating that ICL is a tetramer of identical subunits. The optimum pH was 6.0 in 50 mm phosphate buffer at 30°C. The K_m value of the 35-fold purified ICL for threo-d_s-isocitrate in 50 mm phosphate buffer at pH 6.0 was 0.3 mm. In regulation studies ICL was found to be noncompetitive inhibited by succinate and oxaloacetate. Antibodies against ICL were raised in rabbits. The specificity of the anti-ICL-antibodies was estimated by Ouchterlony tests and by a competitive ELISA on microtiter plates.