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Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast

Titelangaben

Kern, Gunther ; Schülke, Norbert ; Schmid, Franz X. ; Jaenicke, Rainer:
Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.
In: Protein Science. Bd. 1 (1992) Heft 1 . - S. 120-131.
ISSN 1469-896X
DOI: https://doi.org/10.1002/pro.5560010112

Abstract

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.

Weitere Angaben

Publikationsform: Artikel in einer Zeitschrift
Begutachteter Beitrag: Ja
Zusätzliche Informationen: PubMed-ID: 3288623
Institutionen der Universität: Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren > Professur Biochemie - Univ.-Prof. Dr. Franz Xaver Schmid
Fakultäten
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Professur Biochemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren
Titel an der UBT entstanden: Ja
Themengebiete aus DDC: 500 Naturwissenschaften und Mathematik > 540 Chemie
Eingestellt am: 13 Mai 2015 08:16
Letzte Änderung: 05 Sep 2022 11:57
URI: https://eref.uni-bayreuth.de/id/eprint/13443