Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure.
High hydrostatic pressure induced cold inactivation of carboxypeptidase Y. Carboxypeptidase Y was fully active when exposed to subzero temperature at 0.1 MPa; however, the enzyme became inactive when high hydrostatic pressure and subzero temperature were both applied. When the enzyme was treated at pressures higher than 300 MPa and temperatures lower than -5 degrees C, it underwent an irreversible inactivation in which nearly 50% of the alpha-helical structure was lost as judged by circular dichroism spectral analysis. When the applied pressure was limited to below 200 MPa, the cold inactivation process appeared to be reversible. In the presence of reducing agent, this reversible phenomenon, observed at below 200 MPa, diminished to give an inactive enzyme; the agent reduces some of disulfide bridge(s) in an area of the structure that is newly exposed area because of the cold inactivation. Such an area is unavailable if carboxypeptidase Y is in its native conformation. Because all the disulfide bridges in carboxypeptidase Y locate near the active site cleft, it is suggested that the structural destruction, if any, occurs preferentially in this disulfide rich area. A possible mechanism of pressure-dependent cold inactivation of CPY is to destroy the alpha-helix rich region, which creates an hydrophobic environment. This destruction is probably a result of the reallocation of water molecules. Experiments carried out in the presence of denaturing agents (SDS, urea, GdnHCl), salts, glycerol, and sucrose led to a conclusion consistent with the idea of water reallocation.
Kinsho
Toshihiko
T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan.
Ueno
Hiroshi
H
Hayashi
Rikimaru
R
Hashizume
Chieko
C
Kimura
Kunio
K
D002268:Carboxypeptidases / Q000737:chemistry*; D043402:Cathepsin A; D002942:Circular Dichroism; D003080:Cold Temperature; D005990:Glycerol / Q000737:chemistry; D006874:Hydrostatic Pressure; D008623:Mercaptoethanol / Q000737:chemistry; D011489:Protein Denaturation; D020673:Protein Renaturation; D012965:Sodium Chloride / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry
D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D013395:Sucrose; D008623:Mercaptoethanol; D002268:Carboxypeptidases; D043402:Cathepsin A; D005990:Glycerol