tailieunhanh - Báo cáo khoa học: The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure

The heat- and pressure-induced unfolding of the glycosyl-ated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20–75 Corthe pressure range of –700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. | Eur. J. Biochem. 270 4587-4593 2003 FEBS 2003 doi The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure Michiko Kato1. Yasuhiro Sato1. Kumiko Shirai1 Rikimaru Havashi1 Claude Balnv2 and Reinhard Lanae2 1Laboratory of Biomacromolecular Chemistry Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Japan 2INSERM U128 IFR 122 Montpellier France The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and or by their intrinsic fluorescence in the temperature range of 20-75 C or the pressure range of MPa. Under all conditions the precursor form showed a clear two-state transition from a folded to an unfolded state regardless of the presence of the carbohydrate moiety. In contrast the mature form which lacks the propeptide composed of 91 amino acid residues showed more complex behaviour differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains which unfold independently but that procarboxypeptidase Y behaves as a single domain thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding. Keywords carboxypeptidase Y fluorescence spectrometry pressure unfolding procarboxypeptidase Y thermal unfolding. Carboxypeptidase Y CPY a member of the serine carboxypeptidase family is a 61-kDa vacuolar enzyme obtained from Saccharomyces cerevisiae 1 . This enzyme is synthesized in the form of procarboxypeptidase Y pro-CPY and sorted to the vacuole via the Golgi apparatus where it undergoes .