Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases

Abstract

A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.

Figure 1

Western blot following hydrophobic interaction chromatography. A crude extract from 5-d-old dark-grown pumpkin cotyledons was loaded onto an Octyl Sepharose 4FF 1-mL column and protein eluted by decreasing the ammonium sulfate concentration and simultaneously increasing the ethylene glycol concentration. The presence of ACOX activity was tested employing shortchain (hexanoyl-CoA) and medium-/long-chain (decanoyl-CoA and palmitoyl-CoA) substrates. Approximately 20 μL from each even fraction was loaded on each lane. Specific antibodies raised against pumpkin long-chain ACOX (A) and Arabidopsis short-chain ACOX (B) were employed. The positions of the prestained molecular mass standards are indicated on the left.

Figure 2

SDS-PAGE and western-blot analyses of short-chain ACOX on a PhastGel gradient of 10% to 15% following ion-exchange chromatography. Peak fractions (nos. 19–25) following a chromatography on a Mono-S column were subjected to SDS-PAGE and transferred onto a PVDF membrane. Approximately 1 μL from each fraction was loaded per lane. Molecular mass marker positions are indicated on the right. The gel was silver-stained (A) or short-chain ACOX was detected employing antibodies raised against Arabidopsis short-chain ACOX (B).

Figure 3

Determination of the molecular mass of the purified short-chain ACOX by size exclusion chromatography. A sample obtained after Mono-S chromatography was subjected to gel filtration on a Superdex 200 16/60 column. The column was calibrated with thyroglobulin, 669 kD (Thy); ferritin, 440 kD (Fer); catalase, 240 kD (Cat); aldolase, 158 kD (Ald); bovine serum albumin, 67 kD (BSA); ovalbumin, 43 kD (Ova); chymotrypsinogen, 25 kD (Chy); and ribonuclease A, 13.7 kD (Rib). The arrow indicates the elution position of pumpkin short-chain ACOX.

Figure 4

Substrate specificity profiles of purified pumpkin short-chain ACOX. Selectivity for the chain length of the fatty acids acyl-CoA as substrate. All activities were determined using 25 μm acyl-CoA as substrates. The highest activity was arbitrarily set at 100. The se did not exceed 10%.

Figure 5

Activity of the purified short-chain ACOX as a function of substrate concentration in presence or absence of albumin (0.06%, w/v). Enzyme activity was measured with increasing concentration of hexanoyl-CoA with and without albumin. The highest activity was arbitrarily set at 100. The se did not exceed 15%.

Figure 6

Subcellular localization of short-chain ACOX in pumpkin cotyledons. A, An extract from 5-d-old dark-grown pumpkin cotyledons was fractionated by Suc density gradient centrifugation and fractions were tested for enzyme activity: ●, catalase (μmol min⁻¹ mL⁻¹ × 30); ▴, cytochrome c oxidase (nmol min⁻¹ mL⁻¹ × 4); ▵, long-chain ACOX (μmol min⁻¹ mL⁻¹); ○, short-chain ACOX (μmol min⁻¹ mL⁻¹); solid line, Suc concentration. Long-chain ACOX and short-chain ACOX were tested employing palmitoyl-CoA and hexanoyl-CoA, respectively. B, Immunological detection of pumpkin short-chain ACOX by the use of antibodies raised against Arabidopsis short-chain ACOX. Fractions corresponding to mitochondria and peroxisomes are indicated. The position of the prestained molecular mass standard (48.5 kD) is indicated on the right.

Figure 7

Immunoblots showing the cross-reaction of the antiserum raised against the Arabidopsis short-chain ACOX with the corresponding enzyme present in other plants. Crude extracts were obtained from etiolated zucchini cotyledons, etiolated watermelon cotyledons, etiolated castor bean endosperm, etiolated pumpkin cotyledons, Arabidopsis plantlets, etiolated sunflower cotyledons, rocket seedlings, etiolated cucumber cotyledons, maize embryos, broccoli seedlings, etiolated melon cotyledons, etiolated pea cotyledons, and pepper seedlings. Similar amounts of proteins (approximately 100 μg) were loaded on each lane.