The use of subunit hybridization to monitor the reassociation of porcine lactate dehydrogenase after acid dissociation.

Reassociation of tetrameric porcine heart and skeletal muscle lactate dehydrogenases has been previously studied by cross-linking with glutaraldehyde (Bernhardt, G. et al. (1981) Z. Naturforsch. 36c, 772-777; Hermann, R. et al. (1981) Biochemistry 20, 5195-5201). Based on these data, the following kinetic model was proposed for the reconstitution of both isoenzymes: (nontranscribable formula, see text). In the present study, the kinetics of reassociation were determined from the changes of the hybrid patterns obtained upon the mixing of equal amounts of reassociating heart and skeletal muscle lactate dehydrogenases. For a quantitative interpretation of the kinetic data, the dependence of hybrid formation on the conditions of reconstitution was taken into account. Approximately binomial hybrid formation was observed only in a very narrow range of experimental conditions: enzyme concentration greater than 2.7 micrograms/ml, subunit ratio 1:1, temperature range 20-30 degrees C. At a total subunit concentration of 3.4 micrograms/ml (approximately 0.1 micrometers), the reassociation data determined by hybridization were found to be consistent with the proposed kinetic model.