Effect of heavy water and nicotinamide adenine dinucleotide on the sedimentation properties and structure of glyceraldehyde phosphate dehydrogenase

Abstract

The binding of successive nicotinamide adenine dinucleotide (NAD) molecules to the subunits of rabbit muscle glyceraldehyde phosphate dehydrogenase was found by the technique of difference sedimentation to cause an inherent change in the sedimentation coefficient of the protein beyond that attributable to the buoyant weight of the coenzyme. Attempts were made to determine whether the measured changes in sedimentation coefficient were wholly or partially due to shifts in the association-dissociation equilibria between the enzyme and its subunits. The results were equivocal because aqueous preparations of the apoenzyme, produced by charcoal treatment, showed with time increasing amounts of subunits and aggregates larger than tetramers.This spontaneous conversion was accompanied by the loss of potential enzymic activity. Marked stabilization of the apoenzyme was achieved through the use of buffered D20 solutions. The spontaneous inactivation and precipitation observed in aqueous solutions were strikingly eliminated in D20 solutions even at room temperature. In addition, for most preparations of the enzyme in the absence or presence of NAD only tetramers could be detected by sedimentation equilibrium measurements over the concentration range from 0.1 to 3.0 g per 1. This finding was confirmed by differential sedimentation studies which provided direct measurements of the concentration dependence of the sedimentation coefficient. For those preparations showing no evident dissociation in the sedimentation equilibrium experiments the differential sedimentation technique gave similar results for the apoenzyme and enzyme to which different amounts T> X V_abbit muscle glyceraldehyde phosphate dehydrogenase,1 though consisting apparently of four identical polyt From the

The hybridization of glyceraldehyde 3-phosphate dehydrogenases from rabbit muscle and yeast. Kinetics and thermodynamics of the reaction and isolation of the hybrid

“…Slower rates of breakdown of the R3Y and RY3 hybrids in the pH7.8 phosphate buffer would account for the ability to observe these components after electrophoresis in this buffer. This is in accord with the evidence for an increasing tendency for rabbit muscle glyceraldehyde 3-phosphate dehydrogenase to dissociate at increasing pH values in the range pH8 to 10 (Hoagland & Teller, 1969;Smith & Schachman, 1973), and this could result from an increase in the value of the dissociation rate constant as the pH value is raised.…”

A Conformational Change in the Human Major Histocompatibility Complex Protein HLA-DR1 Induced by Peptide Binding

“…The magnitude of this hydrodynamic change is large for a simple ligand-binding reaction. Upon binding a ligand, many other proteins such as citrate synthase, malate synthase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and hexokinase experience changes of only 1-5% in their hydrodynamic parameters, significantly less than the roughly 10-15% seen here (48)(49)(50)(51)(52)(53)(54). Generally, these small changes are caused when the domains of the protein are pulled in closer to each other through interactions triggered by ligand binding.…”

Dynamic Oligomeric Properties

“…In the presence of NAD + or in the presence of both NAD and inorganic phosphate, tetrameric GAPDH appears favored [86,109,110]. The substrate glyceraldehyde plus inorganic phosphate did not have an effect on the equilibrium of the oligomeric forms but glyceraldehyde, NAD and inorganic phosphate together causes a dissociation of the tetramer [1].…”

“…Interesting, they analyzed their data in a model suggesting a dimer by dimer association, stating that they observed dimers, tetramers and octamers. Most support a tetramer-dimer equilibrium [86][87][88]. One laboratory proposes a pentamer of dimers [71].…”