Desai S, Thissen J, Dodd B A, DuBrul E F, Komuniecki R
Mol Biochem Parasitol. 1987 Apr;23(3):203-9. doi: 10.1016/0166-6851(87)90027-2.
Polyclonal antibody was prepared against the pyruvate dehydrogenase complex purified from adult Ascaris suum body wall muscle. The antibody reacted with the E2, X, alpha E1 and beta E1 subunits of the complex in immunoblots of mitochondrial supernatant fractions and homogenates of adult muscle. In addition, the same subunits were observed in immunoblots of homogenates of L3 and L4 ascarid larvae, suggesting that a similar enzyme complex was present in all developmental stages despite their marked differences in energy metabolism. The phosphorylated and dephosphorylated alpha E1 peptides migrated differently during sodium dodecylsulfate polyacrylamide gel electrophoresis and both forms of the enzyme were recognized by the antibody. These results and those obtained with ELISA suggest that both phosphorylated and dephosphorylated forms of the alpha E1 subunit react equally well with the antibody. In immunoblots of adult body wall muscle, the phosphorylated alpha E1 peptide predominated, while immunoblots of L3 larvae contained predominantly the dephosphorylated form. These results reflect the in vivo activity state of the pyruvate dehydrogenase complex in these two stages and suggest that this technique may be useful for determining the activity state of enzyme complex directly from immunoblots of homogenates A. suum and other helminths.
制备了针对从成年猪蛔虫体壁肌肉中纯化的丙酮酸脱氢酶复合物的多克隆抗体。该抗体在线粒体上清液组分和成年肌肉匀浆的免疫印迹中与该复合物的E2、X、α-E1和β-E1亚基发生反应。此外，在L3和L4蛔虫幼虫匀浆的免疫印迹中也观察到了相同的亚基，这表明尽管它们在能量代谢方面存在显著差异，但在所有发育阶段都存在类似的酶复合物。磷酸化和去磷酸化的α-E1肽在十二烷基硫酸钠聚丙烯酰胺凝胶电泳过程中的迁移情况不同，且两种形式的酶都能被该抗体识别。这些结果以及酶联免疫吸附测定（ELISA）的结果表明，α-E1亚基的磷酸化和去磷酸化形式与抗体的反应同样良好。在成年体壁肌肉的免疫印迹中，磷酸化的α-E1肽占主导，而L3幼虫的免疫印迹中主要含有去磷酸化形式。这些结果反映了这两个阶段丙酮酸脱氢酶复合物的体内活性状态，并表明该技术可能有助于直接从猪蛔虫和其他蠕虫匀浆的免疫印迹中确定酶复合物的活性状态。
