Substrate-induced dissociation of glyceraldehyde-phosphate dehydrogenase detected by affinity chromatography. Study of subunit interactions by affinity sorption.
All four Nad-binding domains of the tetrameric apoglyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde 3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) displayed identical affinities (Kd = 4.8 x 10(-5) M) towards the chromophore of Blue Dextran. The enzyme species which contained only the most firmly bound NAD coupled the dye through the remaining free sites with the same affinity as the apoenzyme. The interaction of glyceraldehyde-phosphate dehydrogenase and Blue Dextran-Sepharose was characterized by the aid of an affiinity sorbent batch technique. We found that the apparent dissociation constant varied with enzyme concentration showing preferential binding in diulte solutions. This suggests that the monomeric and/or dimeric form(s) of glyceraldehyde-phosphate dehydrogenase have higher affinity towards the sorbent than the tetramer. The apparent Kd of the enzyme-Blue Dextran-Sepharose complex at low enzyme concentration was similar to the Ki of the competitive inhibition of enzyme activity by free Blue Dextran, i.e. about 10(-6) M. Glyceraldehyde 3-phosphate increased the binding of the enzyme to the immobilized dye in chromatographic experiments and decreased the apparent dissociation constant in the batch system if enzyme solutions of around 10(-6) M were examined. In more dilute solutions glyceraldehyde 3-phosphate had no effect on complex formation. These data suggest that glyceraldehyde 3-phosphate influences the interaction of the enzyme and the immobilized dye, first of all, by shifting the equilibrium of the different enzyme forms towards dissociation.
D000818:Animals; D002846:Chromatography, Affinity; D003911:Dextrans / Q000378:metabolism; D005985:Glyceraldehyde / Q000031:analogs & derivatives*; D005986:Glyceraldehyde 3-Phosphate / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011817:Rabbits; D013057:Spectrum Analysis
D003911:Dextrans; D046911:Macromolecular Substances; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; D005985:Glyceraldehyde; C017080:blue dextran; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases