Identification of the mucin core protein by cell-free translation of messenger RNA from bovine submaxillary glands.
Bovine submaxillary mucin was purified and subjected to chemical deglycosylation by treatment at 20 degrees C with either anhydrous hydrogen fluoride or trifluoromethane sulfonic acid. Virtually all of the sialic acid, galactose, fucose, and over 90% of the N-acetylhexosamines were removed by these treatments. The amino acid compositions of the deglycosylated and native mucins were similar indicating that chemical deglycosylation did not cause significant degradation of the protein. Antiserum specific for the deglycosylated bovine submaxillary mucin was produced by immunization of rabbits with the deglycosylated mucin. RNA was isolated from bovine submaxillary glands by extraction with guanidine hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ mRNA. The poly(A)+ mRNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine, [3H]leucine, [3H]threonine, [3H]proline, or [3H]serine as radiolabel and the translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum. A labeled product of molecular weight 60,000 was present in the immunoprecipitates obtained in the absence but not in the presence of the unlabeled competitor deglycosylated mucin. It is concluded that the primary translation product of the bovine submaxillary gland gene is a 60,000-dalton protein and that the monomer subunit of the mucin is about 170,000. Thus, in the native state the mucin consists of several self-associating subunits.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002474:Cell-Free System; D005779:Immunodiffusion; D009077:Mucins / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012156:Reticulocytes / Q000378:metabolism; D013363:Submandibular Gland / Q000378:metabolism*
D000596:Amino Acids; D002241:Carbohydrates; D009077:Mucins; D012333:RNA, Messenger