Marchat L, Loiseau P M, Petek F
Biologie et Contrôle des Organismes Parasites, Université de Paris-Sud, Châtenay-Malabry, France.
Parasitol Res. 1996;82(8):672-80. doi: 10.1007/s004360050184.
High levels of lactate dehydrogenase (LDH; EC 1. 1. 1. 27) activity have been detected in the filarial worm Molinema dessetae. The two major LDH isoenzymes (LDH1 and LDH2) from female worms were purified by successive chromatography on diethylaminoethyl (DEAE)-Sepharose, carboxymethyl (CM)-Sepharose, and hydroxyapatite columns followed by fast protein liquid chromatography (FPLC)-gel filtration. LDH1 and LDH2 isoenzymes were found to be dimers with subunits of 58 kDa. They had similar properties with regard to substrate and coenzyme affinity. The apparent Michaelis constants (K(m) values; mean +/- SEM, n = 10) were 0.34 +/- 0.04 mM for pyruvate, 0.25 +/- 0.02 mM for reduced nicotinamide adenine dinucleotide (NADH), 2.5 +/- 0.21 mM for lactate, and 0.18 +/- 0.02 mM for NAD, which suggested that pyruvate reduction was the favored reaction. LDH1 and LDH2 were affected by p-chloromercuribenzoate and Hg2+, and such inhibitory effects could be reversed by the addition of thiol compounds (L-cysteine or beta-mercaptoethanol) as observed for mammalian LDH. Oxalate acted as a noncompetitive inhibitor of pyruvate reduction (Ki = 4.7 +/- 0.35 mM; mean +/- SEM, n = 10) and as a competitive inhibitor with lactate (Ki = 2.3 +/- 0.21 mM), whereas oxamate acted as a competitive inhibitor with pyruvate (Ki = 3.3 +/- 0.28 mM) and was noncompetitive with lactate (Ki = 19 +/- 1.2 mM). These substrate analogues exerted similar effects on mammalian LDH, but the inhibition constants were significantly different. The existence of structural and kinetic differences between mammal and filarial LDH isoenzymes prompted us to evaluate them as targets for chemotherapeutic attack.
在丝状线虫德氏莫利线虫中检测到高水平的乳酸脱氢酶（LDH；EC 1.1.1.27）活性。通过依次在二乙氨基乙基（DEAE）-琼脂糖、羧甲基（CM）-琼脂糖和羟基磷灰石柱上进行色谱分离，然后进行快速蛋白质液相色谱（FPLC）-凝胶过滤，从雌虫中纯化出两种主要的LDH同工酶（LDH1和LDH2）。发现LDH1和LDH2同工酶是由58 kDa亚基组成的二聚体。它们在底物和辅酶亲和力方面具有相似的特性。丙酮酸的表观米氏常数（K(m)值；平均值±标准误，n = 10）为0.34±0.04 mM，还原型烟酰胺腺嘌呤二核苷酸（NADH）为0.25±0.02 mM，乳酸为2.5±0.21 mM，NAD为0.18±0.02 mM，这表明丙酮酸还原是更有利的反应。LDH1和LDH2受到对氯汞苯甲酸和Hg2+的影响，并且如在哺乳动物LDH中观察到的那样，这种抑制作用可以通过添加硫醇化合物（L-半胱氨酸或β-巯基乙醇）来逆转。草酸盐作为丙酮酸还原的非竞争性抑制剂（Ki = 4.7±0.35 mM；平均值±标准误，n = 10），并且作为乳酸的竞争性抑制剂（Ki = 2.3±0.21 mM），而草氨酸盐作为丙酮酸的竞争性抑制剂（Ki = 3.3±0.28 mM）并且对乳酸是非竞争性的（Ki = 19±1.2 mM）。这些底物类似物对哺乳动物LDH产生类似的影响，但抑制常数明显不同。哺乳动物和丝状线虫LDH同工酶之间结构和动力学差异的存在促使我们将它们评估为化疗攻击的靶点。
