Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD+ molecules.

1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. Direct binding measurements at 25 degrees C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 micrometer for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD+ bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. Data on the quenching of the protein fluorescence by NAD+ at 15 degrees C at different enzyme concentrations closely fit dissociation constants of 0.028 micrometer for the first two molecules and 0.27 micrometer for the third and fourth molecules.