Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride.
The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.
Liang
S J
SJ
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, China.
Lin
Y Z
YZ
Zhou
J M
JM
Tsou
C L
CL
Wu
P Q
PQ
Zhou
Z K
ZK
D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D008027:Light; D009132:Muscles / Q000201:enzymology; D008121:Nephropidae; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors
D006146:Guanidines; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019791:Guanidine