Effect of coenzymes and temperature on the process of in vitro refolding and reassociation of lactic dehydrogenase isoenzymes

Abstract

Dissociation and deactivation of the H4 pig heart and M4 pig skeletal muscle isoenzymes of lactic dehydrogenase [LDH] in strong denaturants may be reversed with a yield of reactivation up to 100%. The products of reconstitution are indistinguishable from the native enzymes as far as the Km and the Kd for substrate and coenzyme as well as spectral and hydrodynamic properties are concerned. The presence of NAD+ and NADH does not affect either the conformational state of the product of reconstitution, or the kinetics of reactivation, using the pure apoenzymes as a reference. At 20.degree. C the kinetics of reactivation for LDH-M4 in the presence and absence of coenzyme may be quantitatively described by a 2nd-order rate equation (k2 = 23.4 .+-. 2.6 mM-1 s-1) while LDH-H4 is characterized by a uni-bimolecular reaction sequence (k1 = 1.45 .+-. 0.45 .times. 10-3 s-1, k2 = 5 .+-. 1 mM-1 s-1), in agreement with earlier observations. Regarding the influence of temperature on the rate of reactivation no significant anomalies are detectable within the range of 0-25.degree. C. The (apparent) activation energies, taken from the linear Arrhenius plots, are 58 kcal/mol for the association reaction of LDH-M4, and 41 kcal/mol for the transconformation reaction of LDH-H4.