Peroxisomal beta-oxidation system of Candida tropicalis. Purification of a multifunctional protein possessing enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities.

A multifunctional protein from oleate-grown cells of Candida tropicalis has been purified and partially characterized. A simple two-step purification has been developed involving ion-exchange chromatography followed by dye-ligand chromatography on blue Sepharose CL-6B. Homogeneous enzyme with a subunit Mr of 102 000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse-phase high-performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits. Three beta-oxidation activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase, co-purified with this protein. The ratio of the three beta-oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3-hydroxyacyl-CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein. After isopycnic centrifugation of a particulate fraction from oleate-grown cells in a linear sucrose gradient the activities of all individual beta-oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme. The relationship of this multifunctional protein to the two bifunctional beta-oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.

Purification and characterization of 2-enoyl-CoA reductase from bovine liver.

Mitochondrial 2-enoyl-CoA reductase from bovine liver was purified and characterized. A simple three-step purification was developed, involving ion-exchange chromatography to separate the bulk of the NADPH-dependent 2,4-dienoyl-CoA reductase, followed by chromatography on Blue Sepharose and adenosine 2',5'-bisphosphate-Sepharose. Homogeneous enzyme with a subunit Mr of 35 500 is obtained in 35% yield. The Mr of the native enzyme, determined by three different methods, yielded values that suggest that the enzyme is dimeric. NADPH is required as cofactor, and cannot be replaced by NADH. The activity of the purified enzyme towards 2-trans-double bonds in 2-monoene and 2,4-diene structures was investigated. 2-Enoyl-CoA reductase reduced the double bonds in a series of 2-trans-monoenoyl-CoA esters with different chain lengths, but did not exhibit significant activity towards 2-trans-double bonds of 2,4-dienoyl-CoA esters. This result is discussed in the light of analogous observations with enoyl-CoA hydratase.