Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyverpmyces lactis (KIADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmC1 concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KIADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding. (C) 2001 Elsevier Science B.V. All rights reserved.
Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride / Paolo, Sacchetta; Rosalba Di, Rado; Saliola, Michele; Argante, Bozzi; Falcone, Claudio; Carmine Di, Ilio; Filippo, Martini. - In: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY. - ISSN 0167-4838. - 1545:1-2(2001), pp. 238-244. [10.1016/s0167-4838(00)00283-1]
Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride
SALIOLA, Michele;FALCONE, Claudio;
2001
Abstract
Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyverpmyces lactis (KIADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmC1 concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KIADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding. (C) 2001 Elsevier Science B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.