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 Km 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 microM, with a decline at higher concentrations. The enzyme was stable at 70 degrees C for 30 min, but was rapidly inactivated at 90 degrees 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.