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Soluble forms of alpha-D-mannosidases from rat liver. Separation and characterization of two enzymic forms with different substrate specificities.
Grard, T; Saint-Pol, A; Haeuw, J F; Alonso, C; Wieruszeski, J M; Strecker, G; Michalski, J C.
Afiliação
  • Grard T; Laboratoire de Chimie Biologique, (Unité mixte de Recherche du Centre National de la Recherche Scientifique no. 111), Université des Sciences et Technologies de Lille, France.
Eur J Biochem ; 223(1): 99-106, 1994 Jul 01.
Article em En | MEDLINE | ID: mdl-8033914
We have previously reported the substrate specificity of the rat liver cytosolic alpha-D-mannosidase [Haeuw, J. F., Strecker, G., Wieruszeski, J. M., Montreuil, J. & Michalski, J.-C. (1991) Eur. J. Biochem. 202, 1257-1268]. Here, we report the characterization and the purification of this alpha-D-mannosidase and the presence of two soluble forms of alpha-D-mannosidases from rat liver. The cytosolic alpha-D-mannosidase was purified nearly 660-fold with 2.66% recovery to a state approaching homogeneity using: (a) (NH4)2SO4 precipitation; (b) concanavalin-A-Sepharose chromatography; (c) affinity chromatography on a cobalt-chelating Sepharose column; (d) ion-exchange (DEAE-trisacryl M) column chromatography; (e) molecular-size chromatography (Sephacryl S 200). The enzyme was eluted from the final column at an apparent molecular mass of 113 kDa. SDS/PAGE analysis yielded a major protein band at 108 kDa. Moreover, the purification allowed to distinguish two mannosidase activities with different kinetic properties. The first cytosolic activity retained on the cobalt-chelating column was optimally active at neutral pH, was activated by Co2+, was strongly inhibited by swainsonine (Ki = 3.7 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.072 mM). Man9GlcNAc was hydrolysed by the purified enzyme down to a Man5GlcNAc structure, i.e. Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4) GlcNA c, which represents the Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The second activity not retained on the cobalt-chelating column was optimally active at neutral pH, was inhibited by swainsonine (Ki = 28.4 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.633 mM). Man9GlcNAc was broken by this enzymic activity down to Man8GlcNAc and Man7GlcNAc structures. Similitaries with endoplasmic reticulum alpha-D-mannosidase exist and this enzyme could be the cytosolic form of the endoplasmic reticulum alpha-D-mannosidase.
Assuntos
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Base de dados: MEDLINE Assunto principal: Fígado / Manosidases Limite: Animals Idioma: En Revista: Eur J Biochem Ano de publicação: 1994 Tipo de documento: Article País de afiliação: França
Buscar no Google
Base de dados: MEDLINE Assunto principal: Fígado / Manosidases Limite: Animals Idioma: En Revista: Eur J Biochem Ano de publicação: 1994 Tipo de documento: Article País de afiliação: França