title: Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase.
abstract: Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion-exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full-length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F. The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density-gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active-site domain of the enzyme.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Unité 38), Faculté de Médecine, Marseille, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Long
authors.forename: J L
authors.initials: JL
authors.lastname: Franc
authors.forename: J
authors.initials: J
authors.lastname: Kaniewski
authors.forename: J
authors.initials: J
authors.lastname: Lanet
authors.forename: A
authors.initials: A
authors.lastname: Giraud
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004072:Digitonin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006596:Hexosaminidases / Q000737:chemistry; D007453:Iodide Peroxidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000737:chemistry
chemical_list: D011134:Polysaccharides; D007453:Iodide Peroxidase; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D004072:Digitonin