Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

Abstract

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3x10(6)) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15x10(6)). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2x10(5) for mucoprotein A and 2.8x10(4) for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

Isolation and partial characterization of rat gastric mucous glycoprotein

“…Apparently, as also became clear from the carboxymethylcysteine determination, in the unreduced molecules are present disulfide bonds which are cleaved upon reduction. A glycoprotein molecule build up by monomers held together by S-5 bonds would agree with the conclusions of Starkey et al [1] about the structure of pig gastric mucous glycoprotein. 0 value of the reduced glycoproteins 1 (15.7 S) and 2 (11.6 S) were The $20.w in the same order of magnitude as those of the subunits from pig gastric mucous glycoprotein, obtained after reduction [15] and of rat goblet cell mucin [25], an unreduced molecule lacking a subunit structure.…”

“…The relative lower threonine and serine content in the glycoproteins from homogenate fraction I was probably due to a somewhat greater amount of contaminating protein in these unreduced fractions. As was found for pig [1] and dog [2,3], gastric mucus threonine content in glycoproteins 1 and 2 was slightly higher than that of serine. The amount of glycine, glutamic acid and aspartic acid was quite high, as in pig gastric mucus [1].…”

“…Sulphate was determined by the turbidimetric method of Dodgson and Price [19] as modified by Starkey et al [1].…”

“…Much attention has been given to the physicochemical characterization of the glycoproteins that constitute the mucus layer in the porcine [1], the canine [2,3] and the human stomach [4]. Additionally several aspects of glyeoprotein synthesis have been studied.…”

“…For this reason they can be almost completely separated from non-cova]ently bound protein by equflibrium centrifugation in density gradients of highly concentrated cesium salt solutions. This method was used successfully to isolate mucous glycoproteins from pig gastric mucosa [1], from human ovarian cyst fluids [7], from sputum [8] and from cervical mucus [9]. In the experiments described in this paper gastric mucous glycoproteins from rat were isolated on cesium chloride gradients.…”