CHARACTERIZATION OF CDNA CLONES FOR THE 2-METHYL BRANCHED-CHAIN ENOYL-COA REDUCTASE - AN ENZYME INVOLVED IN BRANCHED-CHAIN FATTY-ACID SYNTHESIS IN ANAEROBIC MITOCHONDRIA OF THE PARASITIC NEMATODE ASCARIS-SUUM

The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role i n the reversal of beta-oxidation operating in anaerobic mitochondria o f the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library co nstructed in lambdagt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequence d in both directions. Additional sequence at the 5' end of the mRNA wa s determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a n open reading frame of 1236 nucleotides and a 3'-untranslated sequenc e of 109 nucleotides including a short poly(A) tail 14 nucleotides fro m a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductas e yielded multiple spots with two distinct but overlapping amino-termi nal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of m ammalian acyl-CoA dehydrogenases revealed a high degree of sequence id entity, suggesting that these enzymes may have evolved from a common a ncestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and a dult tissues with antisera that cross-reacted with each of the spots s eparated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revea led a hybridization band of about 1.5 kilobases and also suggested tha t the enzyme was tissue-specific and developmentally regulated in agre ement with the results of the immunoblotting.