Identification, separation, and characterization of acyl-coenzyme A dehydrogenases involved in mitochondrial beta-oxidation in higher plants

Abstract

The existence in higher plants of an additional beta-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of beta-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial beta-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial beta-oxidation in higher plants.

Figure 1

Subcellular localization of palmitoyl-CoA-dependent ACAD activity in embryonic axes from early-germinating sunflower seeds. Crude mitochondria from embryonic axes of early-germinating sunflower seeds were further separated by isopyknic centrifugation on a 20% Percoll gradient. Fractions of 2 mL were collected and assayed for the activity of mitochondrial fumarase (A), palmitoyl-CoA-dependent ACAD (B), peroxisomal ACOX (C), and peroxisomal catalase (D). The scale of volumes from 0% to 100% ranges from the top to the bottom of the Percoll gradient. Separation of organelles and enzyme activity measurements were carried out as described in Methods.

Figure 2

Separation of distinct ACAD by column chromatography on DEAE-Sepharose (A) and hydroxylapatite-HT (B). Proteins from embryonic axes of early-germinating sunflower seeds were fractionated by ammonium sulfate precipitation as described in Methods. Column chromatography and ACAD activity measurements with 50 μm palmitoyl-CoA as the substrate were carried out as described in Methods. A, The resuspended 40% to 60% ammonium sulfate fraction was dialyzed and then loaded onto a DEAE-Sepharose column. Bound proteins were eluted by a linear NaCl gradient from 0 to 0.6 m. B, The pooled fractions from nos. 25 to 48 were concentrated and dialyzed before application to a hydroxylapatite-HT column. Bound proteins were eluted by a linear gradient of phosphate from 0 to 0.5 m. The two peaks of active fractions were pooled separately to give ACAD1 and ACAD2 preparations.

Figure 3

SDS-PAGE analysis of protein fractions in the course of partial purification of ACAD from embryonic axes of early-germinating sunflower seeds. The different protein fractions were obtained as described in Methods and in the legend of Figure 2. Aliquots of the 0% to 40% (lane a, 100 μg of protein) and 40% to 60% (lane b, 100 μg of protein) ammonium sulfate fractions, of the pooled fractions from the DEAE-Sepharose step (lanes c, 100 μg of protein), and of ACAD1 (lanes d, 50 μg of protein) and ACAD2 (lanes e, 12.5 μg of protein) preparations were separated by SDS-PAGE. Proteins were visualized by Coomassie blue staining. The migration of molecular mass markers is given on the right.