Purification and properties of lactate dehydrogenase from liver of Uromastix hardwickii

Lactate dehydrogenase isoenzyme-1 was purified from liver of Uromastix hardwickii using colchicine-Sepharose and heat-inactivation methods. The crude enzyme showed four isoenzymes by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after native AGE and SDS-PAGE corresponding to a molecular weight of 34 kDa. The enzyme did not bind with DEAE-Sepharose at pH 7.2. The optimum pH for forward reaction was 7.5, while for reverse reaction, the maximum activity was at pH 9.5. The K_m values for pyruvate, NADH, lactate and NAD⁺ were 0.105, 0.045, 9.0 and 0.011 mM, respectively. The pyruvate showed maximum activity at about 150 μM and then starts showing inhibition at higher concentration. Pre-heating of enzyme showed that it was stable at 8°C for 30 min and at 100°C it became inactive immediately. Oxalate, glutamate, Cu²⁺, Co²⁺, Mn²⁺, and Mg²⁺ have shown inhibitory effects both for forward- and reverse-reactions. From these properties, we suggest that LDH-1 from Uromastix liver may be quite different from that of other vertebrates.