Purification and characterization of pumpkin long-chain acyl-CoA oxidase |
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| Authors: | Luigi De Bellis Pietro Giuntini Hiroshi Hayashi Makoto Hayashi Mikio Nishimura |
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| Institution: | Dipartimento di Biologia, via Prov.le Lecce-Monteroni, 73100 Lecce, Italy; Dipartimento di Biologia delle Piante Agrarie, via Mariscoglio 34, 56124 Pisa, Italy; Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan |
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| Abstract: | Pumpkin ( Cucurbita sp.) long-chain acyl-CoA oxidase (ACOX) (EC 1.3.3.6) was purified to homogeneity by hydrophobic interaction, hydroxyapatite, affinity, and anion exchange chromatographies. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 72-kDa subunits. The protein is exclusively localized in glyoxysomes. The enzyme catalyzes selectively the oxidation of CoA esters of fatty acids with 12–18 C atoms and exhibits highest activity with C-14 fatty acids, but no activity with isobutyryl-CoA and isovaleryl-CoA (branched chain) or glutaryl-CoA (dicarboxylic). The enzyme is strongly inhibited by high concentrations of palmitoyl-CoA and weakly inhibited by high concentration of myristoyl-CoA. It is also inhibited by Triton X-100 at concentrations above 0.018% (w/v) the critical micellar concentration. The consequences of the substrate inhibition for the evaluation of long-chain ACOX activity in plant tissues are discussed. |
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