Abstract
Some rather large effects of temperature on the kinetics of reactions catalyzed by the rabbit muscle and liver glyceraldehyde-3-phosphate dehydrogenases (GPD) and the results of Kirschner and coworkers (1) on the temperature perturbation of DPN binding by the enzyme from yeast prompted us to examine the temperature dependence of DPN and DPNH binding by the mammalian enzyme and to determine the thermodynamic parameters. In order to cope with the very strong coenzyme binding of the mammalian GPD, we employed the quenching of protein fluorescence as an indicator of complex formation (2) after establishing that the quenching was colinear with binding at the three high affinity sites of the protein. The fourth and weakest binding site does not give an optical signal for ligand addition and must be examined by separat ion methods (3, 4). A set of fluorescence quenching titrations at a series of temperatures is shown in Fig.1. The results at 2.5° are in approximate accord with estimates made by Conway and Koshland (3) at 4° and at much higher protein concentrations by the stepwise removal of bound DPN in a series of prolonged dialyses. The effect of increasing the temperature is to weaken the bind ing and to make the curves progressive I y steeper at their midpoints. Whereas three constants are required to describe the curve at 2.5° , the curve at 36° may be described by a sing le intrinsic binding constant. A similar but slightly less extreme set of curves was obtained in the titration of apo-enzyme with DPNH. The convergence of the three K’s with in-creasing temperature suggest that at still higher temperatures a positive cooperativity might develop, analo-gous to that observed with the yeast enzyme at 40°. However, stability restrictions have prevented us from going much above 36° so that this possibility could not be tested.
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Velick, S.F., Baggott, J.P., Sturrevant, J.M. (1970). Subunit Interactions in Glyceraldehyde-3-Phosphate Dehydrogenase: A Fluorometric and Calorimetric Analysis of DPN Binding as a Function of Temperature. In: Sund, H. (eds) Pyridine Nucleotide-Dependent Dehydrogenases. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-49974-6_20
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DOI: https://doi.org/10.1007/978-3-642-49974-6_20
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