PURIFICATION AND CHARACTERIZATION OF LACTATE-DEHYDROGENASE ISOENZYME-1 AND ISOENZYME-2 FROM MOLINEMA-DESSETAE (NEMATODA, FILARIOIDEA)

High levels of lactate dehydrogenase (LDH; EC 1. 1. 1. 27) activity ha ve been detected in the filarial worm Molinema dessetae. The two major LDH isoenzymes (LDH1 and LDH2) from female worms were purified by suc cessive chromatography on diethylaminoethyl (DEAE)-Sepharose, carboxym ethyl (CM)-Sepharose, and hydroxyapatite columns followed by fast prot ein liquid chromatography (FPLC)-gel filtration. LDH1 and LDH2 isoenzy mes were found to be dimers with subunits of 58 kDa. They had similar properties with regard to substrate and coenzyme affinity. The apparen t 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 adeni ne 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 r eaction. LDH1 and LDH2 were affected by p-chloromercuribenzoate and Hg 2+, and such inhibitory effects could be reversed by the addition of t hiol compounds (L-cysteine or beta-mercaptoethanol) as observed for ma mmalian LDH. Oxalate acted as a noncompetitive inhibitor of pyruvate r eduction (K-i = 4.7 +/- 0.35 mM; mean +/- SEM, n = 10) and as a compet itive inhibitor with lactate (K-i = 2.3 +/- 0.21 mM), whereas oxamate acted as a competitive inhibitor with pyruvate (K-i = 3.3 +/- 0.28 mM) and was noncompetitive with lactate (K-i = 19 +/- 1.2 mM). These subs trate analogues exerted similar effects on mammalian LDH, but the inhi bition constants were significantly different. The existence of struct ural and kinetic differences between mammal and filarial LDH isoenzyme s prompted us to evaluate them as targets for chemotherapeutic attack.