BINDING OF A BURST-PHASE INTERMEDIATE FORMED IN THE FOLDING OF DENATURED D-GLYCERALDEHYDE-3-PHOSPHATE-DEHYDROGENASE BY CHAPERONIN-60 AND 8-ANILINO-1-NAPHTHALENESULPHONIC-ACID

Upon dilution, D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute gu anidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is reactivated only to a limited extent afte r dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensit y of intrinsic fluorescence and binding to 8-anilino-1-naphthalene-sul phonic acid (ANS). This intermediate aggregates with a time lag of a f ew minutes, and the aggregation can be suppressed completely by chaper onin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH r e-activation by Groin suggests that the tetradecameric Groin binds to a dimeric GAPDH folding intermediate. This intermediate can be re-acti vated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably gr eater than that obtained on spontaneous re-activation of the fully den atured enzyme upon dilution. Probing with a fluorescent derivative of NAD(+) shows that this folding intermediate does not have a native con formation at the active site. The similar profiles of the effects of G roin and ANS on the re-activation of GAPDH denatured by different conc entrations of GuHCl suggest that Groin and ANS recognize and bind to t he same folding intermediate, which is similar to the relatively stabl e, partially unfolded, state of the enzyme denatured in 0.5-1.0 M GuHC l. However, the complexes of the intermediate with Groin or ANS appear to be different, in that Groin, but not ANS, suppresses aggregation a nd assists folding in the presence of ATP.