title: Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride.
abstract: Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH 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 GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.
authors.affiliation: Department of Biomedical Sciences, University "G. D'Annunzio", Chieti, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Sacchetta
authors.forename: R
authors.initials: R
authors.lastname: Di Rado
authors.forename: M
authors.initials: M
authors.lastname: Saliola
authors.forename: A
authors.initials: A
authors.lastname: Bozzi
authors.forename: C
authors.initials: C
authors.lastname: Falcone
authors.forename: C
authors.initials: C
authors.lastname: Di Ilio
authors.forename: F
authors.initials: F
authors.lastname: Martini
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000187:drug effects*; D001665:Binding Sites; D002850:Chromatography, Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000187:drug effects*; D019791:Guanidine / Q000494:pharmacology*; D007716:Kluyveromyces / Q000201:enzymology*; D008956:Models, Chemical; D009994:Osmolar Concentration; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
chemical_list: D005656:Fungal Proteins; D000426:Alcohol Dehydrogenase; D019791:Guanidine