|Reference : Contribution of the carbohydrate moiety to conformational stability of the carboxypep...|
|Scientific journals : Article|
|Life sciences : Biochemistry, biophysics & molecular biology|
|Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y high pressure study.|
|Dumoulin, Mireille [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]|
|Ueno, H. [> > > >]|
|Hayashi, R. [> > > >]|
|Balny, C. [> > > >]|
|European Journal of Biochemistry|
|Yes (verified by ORBi)|
|[en] Carboxypeptidases/chemistry/metabolism ; Catalysis ; Cathepsin A ; Enzyme Stability ; Models, Molecular ; Pressure ; Protein Conformation ; Spectrometry, Fluorescence ; Temperature ; Thermodynamics|
|[en] The process of pressure-induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8-anilino-1-naphthalene sulfonic acid binding fluorescences. Pressure-induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8-anilino-1-naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150-500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8-anilino-1-naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule-like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule-like state to a second molten globule-like state. This two-stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule. A similar three-transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure-induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY.|
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