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 mM, respectively. Pyruvate showed maximum activity at about 180 M, with a decline at higher concentrations. The enzyme was stable at 70°C for 30 min, but was rapidly inactivated at 90°C. 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.