MOLECULAR-CLONING, FUNCTIONAL EXPRESSION, AND CHARACTERIZATION OF PYRUVATE-DEHYDROGENASE KINASE FROM ANAEROBIC MUSCLE OF THE PARASITIC NEMATODE ASCARIS-SUUM

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaer obic mitochondrial metabolism of the parasitic nematode Ascaris suum, A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)(+) RNA isolated from adult A. s uum muscle.(2) APDK exhibited significant sequence identity to mammali an PDKs, Nucleotide sequence analysis of the APDK cDNA revealed a 22-n ucleotide spliced leader, characteristic of many nematode mRNAs, a 5'- UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation si gnal. The open reading frame predicted a protein of 399 amino acids wi th a molecular weight of 45,402 that included a putative 18-amino acid leader peptide. Recombinant APDK (rAPDK) was functionally expressed i n Escherichia coli with a his tag at its N-terminus and purified to ap parent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the pre sence of APDK-deficient adult A. suum muscle PDC presumably by the bin ding of APDK to the dihydrolipoyl transacetylase (E2) core of the comp lex. After binding to the core, rAPDK activity was stimulated by eleva ted NADH/NAD(+) and acetyl CoA/CoA ratios within the same ranges as ob served for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional ge ls of the purified adult A. suum muscle PDC. (C) 1998 Academic Press.