Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase.

2,4-Dienoyl-CoA reductase (EC 1.3.1.34) participates in beta-oxidation of (poly)unsaturated enoyl-CoAs and it appears in mammalian mitochondria as two isoforms with molecular masses of 120 and 60 kDa [Hakkola and Hiltunen (1993) Eur. J. Biochem. 215, 199-204]. The 120 kDa isomer is a homotetrameric enzyme, and here we report cDNA cloning of its subunit from human. cDNA clones were isolated by reverse transcriptase-PCR from a fibrosarcoma cell line and by screening from a human liver lambda gt11 cDNA library. The 1128 bp clone contained an open reading frame of 1008 bp encoding a polypeptide of 335 amino acid residues with a predicted molecular mass of 36066 Da. This polypeptide represents the immature monomer of the 120 kDa enzyme, and it contains a predicted N-terminal mitochondrial targeting signal. The amino acid (nucleotide) sequence of human 2,4-dienoyl-CoA reductase shows 82.7% (81.7%) similarity (identity) to the corresponding sequence from the rat. Northern-blot analysis gave a single mRNA species of 1.2 kb in several human tissues, the amounts present in the tissues tested ranking as follows: heart approximately liver approximately pancreas > kidney >> skeletal muscle approximately lung. Immunoblotting of human and rat liver samples with an antibody to the subunit of the rat 120 kDa isoform indicates that the mature human enzyme is larger than its counterpart in the rat. The comparison of amino acid sequences for rat and human enzymes proposes that the difference in the size is 10 amino acid residues. The results show that the rat and human reductases are similar in many characteristics and that the reductase is expressed in human tissues capable of beta-oxidation of fatty acids.

Mitochondrial 2,4-dienoyl-CoA reductases in the rat: differential responses to clofibrate treatment.

The effect of a peroxisome proliferator, clofibrate, on mitochondrial 2,4-dienoyl-CoA reductases was studied in rat liver. The specific activity of reductase measured with 2,4-hexadienoyl-CoA as the substrate increased 2.9-fold in the liver homogenate and 2.5-fold in the mitochondrial extract, whereas acyl-CoA oxidase activity increased 13-fold and delta 3, delta 2-enoyl-CoA isomerase activity, 25-fold in the homogenate. Chromatography of the rat liver homogenate on hydroxylapatite, which separates the mitochondrial isoforms (M(r) 120,000 and M(r) 60,000) showed that the M(r) 60,000 isoform increased 3.5-fold and the M(r) 120,000 isoform 6-fold. When the isoforms were assayed with 2,4-hexadienoyl-CoA and trans-2,cis-4,7,10,13,16,19-docosaheptaenoyl-CoA, the activity ratios of C6 to C22 were 1.5-2.1 for the both isoforms isolated from livers of either control or clofibrate-treated rats. A quantitative immunological experiment with the antibody for the 120,000 reductase in the mitochondrial extracts showed a 6.9-fold increase in the signal, confirming the observation that this isoform is induced more than the other. The mRNA levels of reductase, isomerase, and peroxisomal multifunctional enzyme (MFE) were found to rise in a parallel manner when analyzed by in situ or slot hybridizations, which suggests that the increase was mediated by the same mechanism. Peroxisome proliferators have been shown to increase the mRNA levels of MFE by inducing peroxisome proliferator-activated receptor (PPAR)-mediated expression of the corresponding gene. The short-chain delta 3, delta 2-enoyl-CoA isomerase and the M(r) 120,000 reductase are exceptions among the mitochondrial beta-oxidation proteins, which usually show only a minor response to peroxisome proliferators.

The existence of two mitochondrial isoforms of 2,4-dienoyl-CoA reductase in the rat.

Isoforms of 2,4-dienoyl-CoA reductase (EC 1.3.1.34), which is the key enzyme in the beta-oxidation of fatty acids with double bonds, have been studied in rat heart and liver. Electrofocusing and adsorption chromatography on hydroxyapatite were used to separate the reductase activity in tissue homogenates into two peaks, one peak in each experiment being identified as the previously purified mitochondrial reductase. The novel activity was partially purified from rat liver by means of ammonium sulphate precipitation, anion-exchange chromatography on DEAE-cellulose (DE-52), hydrophobic chromatography on Phenyl-Sepharose and dye-ligand binding chromatography (Blue Sepharose). Taking into account the contribution of the different reductases to the total activity in rat liver, the overall purification for the novel isoform was 1900-fold. Ultracentrifugation on a sucrose gradient gave an M(r) of 50,000 and size-exclusion chromatography on Superdex 200 an M(r) of 60,000. The antibody against the previously characterised reductase did not cross-react with this novel isoform, but the distribution of the activity peaks in heart and liver tissue, and an electrofocusing experiment with isolated mitochondria, both pointed to a mitochondrial origin. The novel reductase was estimated to account for 80% (50%) of the total reductase activity in rat heart (liver) homogenate measured with 2,4-hexadienoyl-CoA. The present results, together with those previously published, suggest that mammals have at least three reductase isoforms: two in mitochondria and a third one in peroxisomes, but the peroxisomal activity has not been characterised so far.

Peroxisomal beta-oxidation of polyunsaturated fatty acids.

Peroxisomes have been shown to play an important role in the oxidative degradation of (poly)unsaturated fatty acids, and contain the enzyme activities needed for the metabolism of double bonds of unsaturated fatty acids in connection with this physiological function. Our understanding of the metabolic pathways and enzyme activities involved in the degradation of unsaturated acyl-CoAs has undergone a re-evaluation recently, and though many open questions still remain significant progress has been made, especially concerning the reactions metabolizing double bonds. The enzyme activities to be discussed here are 2,4-dienoyl-CoA reductase; 3/2-enoyl-CoA isomerase; 2-enoyl-CoA hydratase 2; 5-enoyl-CoA reductase and 3,5/2,4-dienoyl-CoA isomerase. Some of these activities are integral parts of the multifunctional proteins of beta-oxidation systems, which must also be taken into account in this context.

The known purified mammalian 2,4-dienoyl-CoA reductases are mitochondrial isoenzymes.

The aim of this work was to determine the subcellular location of mammalian 2,4-dienoyl-CoA reductase, a key enzyme for degradation of polyunsaturated fatty acids by beta-oxidation. The enzyme was purified according to Kimura et al. (J Biochem 96:1463, 1984), and antibodies were raised in rabbits. Monospecific antibodies were obtained via purification on an affinity column. Immunoblotting of isolated rat liver mitochondria and peroxisomes with the monospecific reductase antibody showed that the antigen was located only in mitochondria. Immunocytochemical experiments with liver tissue, using the protein A-gold labeling technique, confirmed this result. The similarity of their characteristics suggests that the purified reductases described in the literature are the same isoenzyme. Consequently, since the rat enzyme was localized here to the mitochondria, purification and characterization of peroxisomal mammalian reductases remain to be achieved in the future. In addition, a significant induction also of mitochondrial reductase by clofibrate was observed in the immunoblotting experiments.