Purification and characterization of lactate dehydrogenase isoenzymes 1 and 2 from Molinema dessetae (Nematoda:Filarioidea).
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.
Biologie et Contrôle des Organismes Parasites, Université de Paris-Sud, Châtenay-Malabry, France.
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Marchat
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PM
Loiseau
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F
Petek
D000818:Animals; D005260:Female; D005370:Filarioidea / Q000201:enzymology*; D015801:Helminth Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification
D015801:Helminth Proteins; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase