Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio

Lactate dehydrogenase isoenzyme LDH-5 (M4) was purified to homogeneity from the skeletal muscle of lizard Agama stellio stellio as a poikilothermic animal, using colchicine-Sepharose chromatography and heat inactivation. The purified enzyme showed a single band after SDS-PAGE, corresponding to a molecular weight of 36 kD. The K-m values for pyruvate, NADH, lactate, and NAD(+) were 0.020, 0.040, 8.1, and 0.02 m M, respectively Pyruvate showed maximum activity at about 180 muM, with a decline at higher concentrations. The enzyme was stable at 70degreesC for 30 min, but was rapidly inactivated at 90degreesC. The optimum pH for the forward reaction (pyruvate to lactate) was 7.5, and for the reverse reaction (lactate to pyruvate) was 9.2. Oxalate, glutamate, Cu2+ Co2+, Mn2+, and Mg2+ were inhibitory in both forward and reverse reactions.