BINDING AND PROPERTIES OF NAD+ IN GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE FROM LOBSTER-TAIL MUSCLE

1. 1. The extinction coefficient at 280 nm of charcoal-treated lobster-muscle glyceraldehydephosphate dehydrogenase is 0.80 cm2·mg-1. 2. 2. The A280 nm:A260 nm of NAD+-free enzyme was found to be 1.99. 3. 3. Equilibrium dialysis showed that 4 moles of NAD+ may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error (KD < 5·10-9 M), whereas the third and fourth molecules are bound with dissociation constants of respectively 6·10-7 M and 13·10-6 M. 4. 4. The first three NAD+ molecules contribute equally to the absorption at 360 nm, whereas the fourth makes little if any contribution. 5. 5. On mixing NAD+ with the enzyme, the maximum increase of absorption at 360 nm is reached within 3·5 msec, independently of the amount of NAD+ added. This corresponds to a second-order rate constant of more than 1·1010 M-1·sec-1. 6. 6. The reduction of acetyl phosphate by NADH, and its arsenolysis, catalysed by the enzyme, require NAD+. NAD+ has no effect on the esterase activity. © 1969.