Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme

• Published in: Phytochemistry (Oxford) Vol. 62; no. 1; pp. 5 - 11
• Main Authors: Duarte-Vázquez, Miguel A, Garcı́a-Almendárez, Blanca E, Rojo-Domı́nguez, Arturo, Whitaker, John R, Arroyave-Hernández, C, Regalado, Carlos
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 2003; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Brassica napus - enzymology; Brassica napus L. var. purple top white globe; Chemical deglycosylation; Cruciferae; Enzymatic deglycosylation; Enzyme Stability; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycosylation; Hot Temperature; Hydrogen Peroxide - metabolism; Isoenzymes - chemistry; Isoenzymes - metabolism; Kinetics; Metabolism; Oxidation-Reduction; Oxidoreductases; Peroxidase - chemistry; Peroxidase - metabolism; Plant physiology and development; Protein Structure, Secondary; Trypsin - metabolism; Turnip peroxidase; Turnip peroxidase; Cruciferae; Brassica napus L. var. purple top white globe; Enzymatic deglycosylation; Chemical deglycosylation; Purification; Enzyme; Isozyme; Glycoprotein; Mannose; Characterization; Peroxidases; Enzymatic activity; Dicotyledones; Angiospermae; Brassica napus; Spermatophyta; Peroxidase; Oxidoreductases; Kinetics; Galactose
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(02)00456-9

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. A neutral peroxidase isozyme purified from turnip ( Brassica napus L. var. purple top white globe) was partially deglycosylated. The glycoprotein fraction contained (w/w basis): 37% mannose, 31% fucose, 16% galactose, and 15% GlcNAc. The carbohydrate moiety of TP is important for peroxidase activity and stability.