Hymenolepis microstoma: Lactate and malate dehydrogenases of the adult worm

Abstract

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 m M phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K m values for the substrates and cofactors were as follows: pyruvate = 0.51, m M; lactate = 3.8 m M; NADH = 0.011 m M; and NAD = 0.17 m M. NADPH, NADP, or d(−)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate ( p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K m values for substrates and cofactors were as follows: l-malate = 1.09 m M; oxaloacetate = 0.0059 m M; NADH = 0.017 m M; and NAD = 0.180 m M. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 m M). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 m M and 0.5 m M, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.