Equilibrium binding of nicotinamide nucleotides to lactate dehydrogenases.
1. No discontinuities were observed during the continuous titration with NADH of the lactate dehydrogenases of ox muscle, pig heart, pig muscle, rabbit muscle, dogfish muscle or lobster tail muscle. The binding was monitored by either the enhanced fluorescence of bound NADH or the quenched fluorescence of the protein. A single macroscopic dissociation constant, independent of protein concentration, could be used to describe the binding to each enzyme, and there was no need to postulate the involvement of molecular relaxation effects. 2. The affinity for NADH decreases only threefold between pH6 and 8.5. Above pH9 the affinity decreases more rapidly with increasing pH and is consistent with a group of about pK9.5 facilitating binding. Muscle enzymes bind NADH more weakly than does the pig heart enzyme. 3. Increasing temperature and increasing concentrations of ethanol both weaken NADH binding. 4. NADH binding is weakened by increasing ionic strength. NaCl is more effective than similar ionic strengths derived from sodium phosphate or sodium pyrophosphate. 5. Commercial NAD(+) quenches the protein fluorescence of the heart and muscle isoenzymes. Highly purified NAD(+) does not, and its binding was monitored by competition for the NADH-binding sites. A single macroscopic dissociation constant is sufficient to describe NAD(+) binding at the concentrations tested. The dissociation constant is about 0.3mm and is not sensitive to changed ionic strength and to changed pH in the range pH6-8.5.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D008024:Ligands; D008433:Mathematics; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D008121:Nephropidae; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D012754:Sharks; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013696:Temperature; D013816:Thermodynamics
D007527:Isoenzymes; D008024:Ligands; D009243:NAD; D007770:L-Lactate Dehydrogenase