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 anaerobic 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. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide 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 signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed 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 gels of the purified adult A. suum muscle PDC.
Chen
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Department of Biology, University of Toledo, Toledo, Ohio 43606-3390, USA.
Huang
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X
Komuniecki
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PR
Komuniecki
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R
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli; D006801:Humans; D007422:Intestines / Q000469:parasitology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D016366:Open Reading Frames; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012326:RNA Splicing; D018510:RNA, Helminth / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D018510:RNA, Helminth; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases