Trichomonas gallinae: charaterization and regulatory properties of lactic dehydrogenase.

The lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1.1.1.27, LDH)of Trichomonas gallinae was characterized and some of its regulatory properties studied. Electrophoretic analysis, with specific enzymatic staining of crude and dialyzed cell-free extracts and dialyzed ammonium sulfate fractions, all revealed a single band of enzymatic activity suggesting only one molecular form of the enzyme. The pH optima were found to be the following: 7.0 in the pyruvate to lactate direction and 9.0 in the reverse direction. Thermal inactivation studies showed a narrow temperature optimum peaking at 35 C. The K_m values for all four reaction components were determined and found to be: NADH, 70 μm; pyruvate, 88 μm; NAD, 65 μm; and l-lactate, 4.6 mM. T. gallinae LDH was absolutely specific for NAD, NADH, l-lactate, and pyruvate. The enzyme exhibited negative cooperativity, with both NADH and l-lactate, as evidenced by curvilinear Lineweaver-Burk kinetics and Hill coefficients of less than one. Several glycolytic intermediates lowered the K_m of NADH with variable effects on the K_m of pyruvate. The regulation of LDH by glycolytic intermediates is discussed.