caa a22 4500 475744551 CHVBK 20180406123509.0 cr unu---uuuuu 170329e20000701xx s 000 0 eng 10.1023/A:1026483313258 doi (NATIONALLICENCE)springer-10.1023/A:1026483313258 Salt-Induced Folding of a Rabbit Muscle Pyruvate Kinase Intermediate at Alkaline pH [Elektronische Daten] [F. Edwin, M. Jagannadham] The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na2SO4 to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation. Plenum Publishing Corporation, 2000 Pyruvate kinase nationallicence alkaline denaturation nationallicence guanidine hydrochloride nationallicence ANS binding nationallicence circular dichroism nationallicence intermediate state nationallicence Edwin F. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India aut Jagannadham M. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 361-371 0277-8033 19:5<361 2000 19 10930 https://doi.org/10.1023/A:1026483313258 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026483313258 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Edwin F. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India aut NATIONALLICENCE 700 1- Jagannadham M. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/5(2000-07-01), 361-371 0277-8033 19:5<361 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer