Kinetic Properties of Lactate Dehydrogenase from Trout Muscle

The kinetics of pyruvate reduction by LDH isozymes in the presence of NADH is said to be an ordered bisubstrate reaction characterized by Michaelian concentration dependence (Borgmann et al., 1975). If the reaction is ordered, no cooperativity can occur between the two substrates (NADH and pyruvate) (Neet, 1995). Experiments with fish LDH showed, however, that this enzyme might display two types of saturation profile, hyperbolic and sigmoidal, depending on the pH in the reaction medium. At pH 9, for example, the initial velocity of pyruvate reduction by epaxial muscle LDH from goldfish showed a sigmoidal dependence upon substrate concentration, whereas at pH 8 the kinetics were of the hyperbolic type (Hochachka, 1965). Our experiments showed, in addition, that LDH from trout muscle (Onchorhynchus mykiss) could display both types of saturation profile at one and the same pH, but in the presence of different concentrations of the LDH preparation. Rainbow trout (Onchorhyncus mykiss) were obtained in October from the Goc Fishpond of the School of Forestry, University of Belgrade, Serbia. White skeletal muscle was immediately excised and kept in liquid nitrogen until use. After thawing, muscle samples were deprived of skin and spine, cut into small pieces, and homogenized in 5 volumes (w/v) of Tris-EDTA buffer. The homogenate was centrifuged for 10 min at 10 000× g. Partial purification with ammonium sulfate was performed according to Place and Powers, 1984. After centrifugation, solid ammonium sulfate was added to the supernatant to give 30% saturation at 0oC. The suspension was stirred for 1 hour and centrifuged for 30 min at 10 000× g. The pellet was discarded, and the supernatant brought to 70% saturation. The suspension was then stirred again for 1 h at 0oC, and centrifuged as already described. This time, the supernatant was discarded and the pellet redissolved in Tris-EDTA buffer and dialyzed overnight at 4oC against four 1liter replacements of the same buffer. The concentration of protein, determined by the method of Lowry (Lowry et al., 1951), amounted to 5.3 mg/ml of the final enzyme suspension.