title: Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme.
abstract: A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.
authors.affiliation: Departamento de Investigación y Posgrado en Alimentos, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro, C. U. Cerro de Las Campanas S/N, Querétaro, Qro. 76010, Mexico.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Duarte-Vázquez
authors.forename: Blanca E
authors.initials: BE
authors.lastname: García-Almendárez
authors.forename: Arturo
authors.initials: A
authors.lastname: Rojo-Domínguez
authors.forename: John R
authors.initials: JR
authors.lastname: Whitaker
authors.forename: C
authors.initials: C
authors.lastname: Arroyave-Hernández
authors.forename: Carlos
authors.initials: C
authors.lastname: Regalado
mesh_terms: D029688:Brassica napus / Q000201:enzymology*; D004795:Enzyme Stability; D006031:Glycosylation; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D014357:Trypsin / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D006861:Hydrogen Peroxide; D009195:Peroxidase; D014357:Trypsin