EXPRESSION OF PYRUVATE-DEHYDROGENASE ISOFORMS DURING THE AEROBIC ANAEROBIC TRANSITION IN THE DEVELOPMENT OF THE PARASITIC NEMATODE ASCARIS-SUUM - ALTERED STOICHIOMETRY OF PHOSPHORYLATION/INACTIVATION/

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaer obic metabolism of the parasitic nematode Ascaris suum. Two isoforms o f the a-subunit of pyruvate dehydrogenase (E1) have been identified: a lpha I is most abundant in anaerobic adult muscle and alpha II in aero bic larvae. Both isoforms have been expressed as alpha(2) beta(2) tetr amers with a muscle-specific beta-subunit, purified to apparent homoge neity, reconstituted with E1-deficient adult A. suum muscle PDC, and a ssayed for PDC and E1 kinase activity. Recombinant alpha II is a poor substrate for the adult E1 kinase, bit its stoichiometry of phosphoryl ation/inactivation is similar to that reported for the human E1. Initi ally, inactivation parallels the incorporation of about 1 mol P-32/mol E1 and at maximal phosphorylation about 2.4 mol P-32/moI E1 is incorp orated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivat ion. To examine this altered pattern of phosphorylation, the two phosp horylation sites in each E1 alpha subunit of the r alpha I (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 t han site 2, but the phosphorylation of either site resulted in inactiv ation, and the phosphorylation of only a single E1 alpha subunit of th e tetramer was necessary for inactivation. However, both E1 alpha subu nits of the tetramer were phosphorylated, based on the incorporation o f about 3.5 mol P-32/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha subunits during SDS-PAGE. These obse rvations suggest that the regulation of both E1 isoforms is modified t o maintain PDC activity during the transition to anaerobiosis. (C) 199 8 Academic Press.