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 anaerobic metabolism of the parasitic nematode Ascaris suum. Two isoforms of the alpha-subunit of pyruvate dehydrogenase (E1) have been identified: alpha I is most abundant in anaerobic adult muscle and alpha II in aerobic larvae. Both isoforms have been expressed as alpha 2 beta 2 tetramers with a muscle-specific beta-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adult A. suum muscle PDC, and assayed for PDC and E1 kinase activity. Recombinant alpha II 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 mol 32P/mol E1 and at maximal phosphorylation about 2.4 32P/mol E1 is incorporated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation 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 than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1 alpha subunit of the tetramer was necessary for inactivation. However, both E1 alpha subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol 32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha 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.
Huang
Y J
YJ
Department of Biology, University of Toledo, Ohio 43606-3390, USA.
Walker
D
D
Chen
W
W
Klingbeil
M
M
Komuniecki
R
R
D000332:Aerobiosis / Q000502:physiology; D000693:Anaerobiosis / Q000502:physiology; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology* / Q000254:growth & development; D001665:Binding Sites / Q000502:physiology; D004795:Enzyme Stability / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015801:Helminth Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics
D015801:Helminth Proteins; D007527:Isoenzymes; D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D011994:Recombinant Proteins; D030481:Pyruvate Dehydrogenase (Lipoamide); C065640:pyruvate dehydrogenase E1alpha subunit