Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone.
We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.
Department of Cell Biology, State University of Utrecht, School of Medicine, The Netherlands.
J
J
Dekker
W M
WM
Van Beurden-Lamers
A
A
Oprins
G J
GJ
Strous
D000818:Animals; D005753:Gastric Mucosa / Q000032:analysis*; D006023:Glycoproteins / Q000302:isolation & purification* / Q000648:ultrastructure; D008854:Microscopy, Electron; D051381:Rats
D006023:Glycoproteins; C047874:gastric mucus glycoproteins