A Bifunctional Enzyme from Glyoxysomes

Abstract

Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase copurified when extracts from cotyledons of 5 day old cucumber seedlings were fractionated by acetone precipitation, ion-exchange chromatography on carboxymethyl-(CM)-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM-cellulose, or blue-dextran-Sepharose, respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein. Further characterization of the protein revealed that the 2 enzyme activities are contained in a single peptide chain of MW 75,000. An extremely alkaline isoelectric point of pH 9.8 was determined. The bifunctional protein was localized in glyoxysomes which were the exclusive site of .beta.-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle''s membrane.