PHYSIOLOGIA PLANTARUM 106: 170 – 176. 1999 Copyright © Physiologia Plantarum 1999 ISSN 0031-9317 Printed in Ireland —all rights resered Purification and characterization of pumpkin long-chain acyl-CoA oxidase Luigi De Bellis a,b, *, Pietro Giuntini b , Hiroshi Hayashi c , Makoto Hayashi c and Mikio Nishimura c a Dipartimento di Biologia, ia Pro. le Lecce -Monteroni, 73100 Lecce, Italy b Dipartimento di Biologia delle Piante Agrarie, ia Mariscoglio 34, 56124 Pisa, Italy c Department of Cell Biology, National Institute for Basic Biology, Okazaki 444 -8585, Japan *Corresponding author, e -mail: debelli@ilenic.unile.it Received 12 January 1999; revised 17 March 1999 Pumpkin (Cucurbita sp.) long-chain acyl-CoA oxidase isovaleryl-CoA (branched chain) or glutaryl-CoA (dicar- (ACOX) (EC 1.3.3.6) was purified to homogeneity by hydro- boxylic). The enzyme is strongly inhibited by high concentra- phobic interaction, hydroxyapatite, affinity, and anion ex- tions of palmitoyl-CoA and weakly inhibited by high change chromatographies. The purified isoenzyme is a dimeric concentration of myristoyl-CoA. It is also inhibited by Triton protein, consisting of two apparently identical 72-kDa sub- X-100 at concentrations above 0.018% (w/v) the critical mi- cellar concentration. The consequences of the substrate inhibi- units. The protein is exclusively localized in glyoxysomes. The enzyme catalyzes selectively the oxidation of CoA esters of tion for the evaluation of long-chain ACOX activity in plant fatty acids with 12–18 C atoms and exhibits highest activity tissues are discussed. with C-14 fatty acids, but no activity with isobutyryl-CoA and Introduction Acyl-CoA oxidases (ACOXs, EC 1.3.3.6) are flavoproteins which catalyze the initial step in each cycle of the perox- isomal  -oxidation, the conversion of acyl-CoA to trans - 2-enoyl-CoA, transferring electrons to molecular oxygen to produce hydrogen peroxide. In mammalian cells both mitochondria and peroxisomes host an active and well-characterized  -oxidation path- way. In mitochondria the first step of the pathway is cata- lyzed by acyl-CoA dehydrogenases which transfer electrons to an electron transfer flavoprotein and then to the mitochondrial respiratory chain. Four acyl-CoA dehy- drogenases which vary in their specificity for acyl-chain length are present allowing mammalian mitochondria to perform the  -oxidation of the entire acyl-CoA esters (Eaton et al. 1996). In contrast, peroxisomal  -oxidation can metabolize long-chain acyl-CoA esters only partly be- cause the three peroxisomal ACOXs (palmitoyl-CoA oxi- dase, pristanoyl-CoA oxidase, and trihydroxycopros- tanoyl-CoA oxidase) show a very low affinity for short- chain acyl-CoAs (Van Veldhoven et al. 1992). The affinity of the plant peroxisomal ACOXs for both long- and short-chain acyl-CoAs (Gerhardt 1985, Hook et al. 1995) permits the plant peroxisomes to degrade the fatty acids completely. In addition, plant peroxisomes are capable of  -oxidizing a variety of substrates: CoA esters of straight-chain and unsaturated fatty acids; esters of un- common fatty acids such as ricinoleic acid; and branched- chain 2-oxo acids (Gerhardt 1992). Several papers of Thomas and coworkers (e.g. Wood et al. 1986, Miernyk et al. 1991) and Dieuaide et al. (1993) have indicated a double location of  -oxidation also in plants. More re- cently, Anderson et al. (1998) showed that a branched- chain acyl-CoA dehydrogenase is present in soybean mitochondria. However, none of the plant mitochondrial  -oxidation enzymes has been purified and/or character- ized. Some plant ACOXs have been purified to apparent ho- mogeneity: long-chain ACOX from cucumber cotyledons (Kirsh et al. 1986) and medium- and short-chain ACOX from maize plantlets (Hook et al. 1996). They are charac- terized by different molecular masses (from 15 kDa for short-chain ACOX to 72 kDa for long-chain ACOX) of the subunits. So far, the activity on branched-chain or dicarboxylic acyl-CoAs has not been analyzed. Abbreiations – ACOX, acyl-CoA oxidase; FAD, flavin adenine dinucleotide; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; SHAM, salicylhydroxamate. Physiol. Plant. 106, 1999 170