Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod (G. morhua) using a 96-well microplate technique.
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.
Zakhartsev
Maxim V
MV
Department of Biology, University of Antwerp-RUCA, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. m.zakhartsev@iu-bremen.de
Pörtner
Hans O
HO
Blust
Ronny
R
D000818:Animals; D003080:Cold Temperature; D048389:Gadus morhua; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000201:enzymology*; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods*; D013816:Thermodynamics
D007770:L-Lactate Dehydrogenase