Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons 1

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purified from glyoxysomes of germinating pumpkin cotyledons. Molecular weights of their subunits were as follows: malate synthase, 60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase, 72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were exclusively localized in glyoxysomes, whereas malate dehydrogenase and citrate synthase activities were found in both glyoxysomes and mitochondria. Monospecific antibodies against malate dehydrogenase and citrate synthase inhibited their activities present in glyoxysomes but in mitochondria. Immunocytochemical analysis using the protein A-gold technique combined with Lowicryl K4M embedding showed that the antigenic sites for these enzymes were found exclusively in glyoxysomes. These data indicates that malate dehydrogenase and citrate synthase present in glyoxysomes are immunologically different from those in mitochondria, respectively.