Mottram J C, Coombs G H
Biochim Biophys Acta. 1985 Mar 1;827(3):310-9. doi: 10.1016/0167-4838(85)90216-x.
The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.
墨西哥利什曼原虫无鞭毛体苹果酸脱氢酶（L-苹果酸：NAD⁺氧化还原酶，EC 1.1.1.37）和磷酸烯醇丙酮酸羧激酶（ATP：草酰乙酸羧基裂解酶（转磷酸化），EC 4.1.1.49）的颗粒活性，通过使用苯基琼脂糖凝胶CL-4B的疏水相互作用色谱法、使用5'-AMP琼脂糖凝胶4B的亲和色谱法以及使用葡聚糖凝胶G-100的凝胶过滤法，已被纯化至表观电泳均一性。苹果酸脱氢酶总体上纯化了150倍，最终比活性为1230单位/毫克蛋白质，回收率为63%。磷酸烯醇丙酮酸羧激酶纯化了132倍，最终比活性为30.3单位/毫克蛋白质，回收率为20%。通过凝胶过滤和SDS-凝胶电泳测定的苹果酸脱氢酶分子量分别为39800和33300，磷酸烯醇丙酮酸羧激酶分子量分别为63100和65100。在草酰乙酸还原方向上测定苹果酸脱氢酶的动力学研究表明，Km（NADH）为41微摩尔，Km（草酰乙酸）为39微摩尔。对于苹果酸氧化，Km（苹果酸）为3.6毫摩尔，Km（NAD）为0.79毫摩尔。草酰乙酸在浓度大于0.83毫摩尔时表现出底物抑制作用，并且发现苹果酸在高浓度时是产物抑制剂。然而，许多糖酵解中间产物和辅因子对酶活性没有影响，这表明苹果酸脱氢酶不是墨西哥利什曼原虫中的主要调节酶。结果表明，这些墨西哥利什曼原虫无鞭毛体酶在几个方面与其哺乳动物对应物相似；尽管如此，它们在寄生虫中明显的重要性和独特的亚细胞组织使其成为化学治疗攻击的潜在靶点。
