RESUMEN
Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.
Asunto(s)
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Extractos Vegetales/química , Arabidopsis/química , Arabidopsis/enzimología , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Plantas/metabolismo , Polisacáridos/metabolismoRESUMEN
Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.