Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase.
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.
Long
Y
Y
Institut National de la Santé et de la Recherche Médicale (Unité 38), Faculté de Médecine, Marseille, France.
Franc
J L
JL
Kaniewski
J
J
Lanet
J
J
Giraud
A
A
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
D011134:Polysaccharides; D007453:Iodide Peroxidase; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D004072:Digitonin