Expression of Pyruvate Dehydrogenase Isoforms during the Aerobic/Anaerobic Transition in the Development of the Parasitic NematodeAscaris suum:Altered Stoichiometry of Phosphorylation/Inactivation

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic metabolism of the parasitic nematodeAscaris suum.Two isoforms of the α-subunit of pyruvate dehydrogenase (E1) have been identified: αI is most abundant in anaerobic adult muscle and αII in aerobic larvae. Both isoforms have been expressed as α2β2tetramers with a muscle-specific β-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adultA. suummuscle PDC, and assayed for PDC and E1 kinase activity. Recombinant αII is a poor substrate for the adult E1 kinase, but its stoichiometry of phosphorylation/inactivation is similar to that reported for the human E1. Initially, inactivation parallels the incorporation of about 1 mol32P/mol E1 and at maximal phosphorylation about 2.4 mol32P/mol E1 is incorporated. In contrast, recombinant αI (rαI) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation sites in each E1α subunit of the rαI (site 1 and site 2) were changed either individually or together from Ser to Ala by site-directed mutagenesis. Site 1 was phosphorylated more rapidly than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1α subunit of the tetramer was necessary for inactivation. However, both E1α subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1α subunits during SDS–PAGE. These observations suggest that the regulation of both E1 isoforms is modified to maintain PDC activity during the transition to anaerobiosis.