Purification and immunofluorescent localization of rat submandibular mucin.
Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S(0) (20,w) 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1x10(6). Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D005455:Fluorescent Antibody Technique; D007122:Immunoelectrophoresis; D008297:Male; D009077:Mucins / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013363:Submandibular Gland / Q000032:analysis*
D000596:Amino Acids; D002241:Carbohydrates; D009077:Mucins