Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod (G....

Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0-40 degrees C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 degrees C with high stability and accuracy in any data acquisition mode. At 4 degrees C the accuracy of the temperature control was +/-0.1 degrees C and temperature homogeneity across the microplate was +/-0.3 degrees C. As examples, analyses of the temperature dependence of Michaelis-Menten (K'(PYR)(m) and substrate inhibition (K'(PYR)(si) constants for pyruvate, of the maximal rate of reaction (V'(max), of the apparent Arrhenius activation energy (E(A), and of the Gibbs free-energy change (deltaG) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 degrees C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.