Isolation and Characterization of Citrate Synthase Isozymes from Castor Bean Endosperm

Abstract

By cellulose acetate gel electrophoresis and DEAE-cellulose column chromatography, two distinct citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4. 1. 3. 7] isozymes were isolated from castor bean endosperm. Mitochondria and glyoxysomes were isolated by sucrose density gradient centrifugation. The first and second peaks of citrate synthase activities of DEAE-cellulose column chromatography corresponded electrophoretically with the bands obtained from the glyoxysomes and mitochondria respectively. The optimum pH values for the two isozymes were 8.0 to 8.1 similarly, but kinetic properties were different. The Michaelis constants for acetyl-CoA and oxaloacetate of the mitochondria) citrate synthase (M-CS) at pH 8.0 were 3.4×10^-5_M and 0.83×10^-5_M, respectively. Those of the glyoxysomal citrate synthase (G-CS) were 8.5×10^-5_M and 4.5×10^-5_M, respectively. Metabolites such as citrate, glyoxylate, and succinate partially inhibited the isozymes. The degree of inhibition was stronger with M-CS than with G-CS.