Song H, Komuniecki R
Department of Biology, University of Toledo, Ohio 43606-3390.
J Biol Chem. 1994 Dec 16;269(50):31573-8.
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.
丙酮酸脱氢酶（PDHb）磷酸酶已从成年寄生线虫猪蛔虫的线粒体中纯化至表观均一。通过SDS-聚丙烯酰胺凝胶电泳判断，该酶是一个由89 kDa和50 kDa组成的异二聚体。它似乎与其底物丙酮酸脱氢酶（E1）共同纯化，并且在0.5 M NaCl存在下通过Superose 12柱层析可将它们分离。磷酸酶活性绝对依赖于Mg2+，表观Km约为4 mM。与其他来源的PDHb磷酸酶不同，蛔虫的这种酶不受Ca2+或精胺刺激，但受L-苹果酸刺激，L-苹果酸是猪蛔虫主要的线粒体底物。L-苹果酸对分离的[32P]E1的去磷酸化没有影响，但它使磷酸酶对32P-丙酮酸脱氢酶复合物或重组复合物中[32P]E1的表观Km降低约4至6倍，这表明二氢硫辛酰胺转乙酰酶（E2）核心对于苹果酸激活是必需的。分离的猪蛔虫肌肉线粒体中丙酮酸脱氢酶复合物（PDC）的活性显著高于其他线粒体报道的活性，并且大多数PDC似乎处于磷酸化的无活性状态。在无底物且存在解偶联剂的情况下孵育完整的进行磷酸化的猪蛔虫肌肉线粒体不会导致PDC活性的激活。相反，在苹果酸加Mg2+中孵育会显著增加PDC活性。这些结果与需氧线粒体报道的结果明显不同，表明这些厌氧细胞器中PDC活性的调节与其哺乳动物宿主的调节有显著差异。
