Novel regulation of pyruvate dehydrogenase phosphatase purified from anaerobic muscle mitochondria of the adult parasitic nematode, Ascaris suum.
Pyruvate dehydrogenase (PDHb) phosphatase has been purified to apparent homogeneity from mitochondria of the adult parasitic nematode, Ascaris suum. The enzyme is a heterodimer of 89 and 50 kDa, as judged by SDS-polyacrylamide gel electrophoresis. It appeared to copurify with its substrate, pyruvate dehydrogenase (E1) and could be separated by chromatography on Superose 12 in the presence of 0.5 M NaCl. Phosphatase activity was absolutely dependent on Mg2+, with an apparent Km of about 4 mM. In contrast to PDHb phosphatases from other sources, the ascarid enzyme was not stimulated by Ca2+ or spermine, but it was stimulated by L-malate, the major mitochondrial substrate in A. suum. L-Malate had no effect on the dephosphorylation of isolated [32P]E1, but it decreased the apparent Km of the phosphatase for 32P-pyruvate dehydrogenase complex or [32P]E1 in the reconstituted complex about 4-6-fold, suggesting that the dihydrolipoyl transacetylase (E2) core was necessary for malate activation. The activity of the pyruvate dehydrogenase complex (PDC) in isolated A. suum muscle mitochondria was significantly greater than that reported for other mitochondria, and the majority of the PDC appeared to be in the phosphorylated inactive state. Incubation of intact phosphorylating A. suum muscle mitochondria in the absence of substrate and the presence of an uncoupler did not lead to an activation of PDC activity. In contrast, incubation in malate plus Mg2+ markedly increased PDC activity. These results contrast markedly with those reported for aerobic mitochondria and suggest that the regulation of PDC activity in these anaerobic organelles differs significantly from that of their mammalian hosts.
Song
H
H
Department of Biology, University of Toledo, Ohio 43606-3390.
Komuniecki
R
R
D000693:Anaerobiosis; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism*
D008293:Malates; D010750:Phosphoproteins; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase