caa a22 4500 465734499 CHVBK 20180323111751.0 cr unu---uuuuu 170327e19900701xx s 000 0 eng 10.1007/BF01073372 doi (NATIONALLICENCE)springer-10.1007/BF01073372 Optimized deglycosylation of glycoproteins by peptide-N4-( N -acetyl-β-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum [Elektronische Daten] [R Nuck, M Zimmermann, D Sauvageot, D Josić, W Reutter] Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (PNGase F) fromFlavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N(asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with humanα 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of humanα 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10. Glycoconjugate Journal AB, 1990 Nuck R. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut Zimmermann M. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut Sauvageot D. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut Josić D. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut Reutter W. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut Glycoconjugate Journal Kluwer Academic Publishers 7/4(1990-07-01), 279-286 0282-0080 7:4<279 1990 7 10719 https://doi.org/10.1007/BF01073372 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF01073372 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nuck R. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut NATIONALLICENCE 700 1- Zimmermann M. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut NATIONALLICENCE 700 1- Sauvageot D. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut NATIONALLICENCE 700 1- Josić D. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut NATIONALLICENCE 700 1- Reutter W. Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-1000, Berlin 33 (Dahlem), Germany aut NATIONALLICENCE 773 0- Glycoconjugate Journal Kluwer Academic Publishers 7/4(1990-07-01), 279-286 0282-0080 7:4<279 1990 7 10719 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer