A bifunctional enzyme from glyoxysomes. Purification of a protein possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities.

1. 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 CM-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. 2. 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. 3. Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined. 4. The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of beta-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.