Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases.
1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.
D000429:Alcohol Oxidoreductases; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005453:Fluorescence; D005969:Glutamate Dehydrogenase; D005993:Glycerolphosphate Dehydrogenase; D006736:Horses; D008024:Ligands; D008099:Liver / Q000201:enzymology; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D009249:NADP; D009492:Neurospora crassa / Q000201:enzymology; D010088:Oxidoreductases
D008024:Ligands; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D005993:Glycerolphosphate Dehydrogenase; D005969:Glutamate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases