Effects of hydrogen peroxide, mild trypsin digestion and partial reduction on rat intestinal mucin and its disulphide-bound 118 kDa glycoprotein.
The role of the disulphide-bound 118 kDa glycoprotein of rat intestinal mucin is unknown, although it has been proposed to serve as a 'link' component for the mucin monomers. The present studies investigated release or destruction of the 118 kDa glycoprotein (monitored by gel electrophoresis and Western-blot analysis) during progressive breakdown of the mucin polymer (assessed by Sepharose 2B chromatography). H2O2 gradually destroyed the 118 kDa glycoprotein and dissociated the mucin polymer into components of similar size to the monomers. After 3 h, mucin samples contained almost no 118 kDa glycoprotein or its breakdown products, but 50% of the mucin was still eluted in the void volume of a Sepharose 2B column. Although mild trypsinolysis had little effect on the Sepharose 2B elution profile of the mucin, the 118 kDa glycoprotein was completely cleaved into 54-56 kDa and 60-66 kDa fragments which remained disulphide-bound to the high-molecular-mass mucin. Increasing levels of thiol reduction resulted in progressive loss of disulphide bonds, release of the 118 kDa glycoprotein and depolymerization of the mucin. Although approx. 40% of the mucin in partially reduced samples was recovered in the Sepharose 2B void volume, this material contained no 118 kDa glycoprotein and apparently consisted of disulphide-bound mucin monomers. Thus the 118 kDa glycoprotein may be destroyed by H2O2, extensively cleaved by trypsin or released by reduction without completely dissociating the mucin into monomers. Therefore the 118 kDa glycoprotein may not function as a 'link' component for all of the mucin monomers in the native polymer.
Mantle
M
M
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects*; D008297:Male; D008970:Molecular Weight; D009077:Mucins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D006023:Glycoproteins; D009077:Mucins; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D014357:Trypsin