Isolation and characterization of canine submaxillary mucin

Abstract

The isolation and properties of mucin from submaxillary glands of a number of species of animals have been reported (reviewed by Pigman & Gottschalk, 1966).The present communication describes a method for preparation of purified canine submaxillary mucin involving gel filtration on Sephadex G-200 with an additional fractionation step using Bio-Gel CM-100. This method yields a native and highly purified material from submaxillary glands and from submaxillary secretions. The composition of the purified mucin indicates that it is a sulphated glycoprotein containing 35 % protein, 61 % carbohydrate and 3.8% sulphate. The findings extend previous work on canine submaxillary mucin reported by Hashimoto & Pigman (1962).Canine submaxillary glands either were obtained from dogs under anaesthesia and immediately frozen in liquid N2, or were obtained frozen from Pel-Freeze Inc. (Rogers, Ark., U.S.A.). The glands were decapsulated, freed from connective tissue and fat, cut into small pieces and extracted with 2vol. of ice-cold distilled water containing NaN3 (5mM) for 24h at 4°C (extract 1). Extracts 2 and 3 were obtained by repeating the extraction twice with 1 vol. of water for 48h. The extracts were combined and dialysed overnight in the cold against 0.1 M-sodium acetate buffer, pH 3.9, and a small precipitate was removed by centrifugation.Then 80ml of the combined dialysed supernatant, containing 610mg of protein, was applied to a Sephadex G-200 column (5cm x 81cm) previously equilibrated with 0.1 M-sodium acetate buffer, pH3.9. Elution was carried out with the same buffer at a flow rate of 36ml/h. Fractions of volume 20ml were collected, and samples were analysed for protein by measuring the absorbance at 280nm, for hexoses by the orcinol method (Winzler, 1955) and for sialic acid by the direct Ehrlich method (Werner & Odin, 1952). Virtually all of the sialic acid, 93 % of the hexose and 43 % of the protein were eluted in the void volume. The remaining protein was eluted in two retarded peaks, one of which contained some hexose. Recoveries of sialic acid, hexose and protein were greater than 95 %. For large-scale preparations 300ml (2.29g of protein) was applied to a column of larger size (9.7cm x 105 cm). The fractions constituting the Vol. 128

Purification and immunofluorescent localization of rat submandibular mucin

“…Calculations based on estimates of protein (Lowry method) and sugar (g.l.c. analysis) content showed that the RSM isolated in our studies consisted of 63% (w/w) carbohydrate, a value within the usual range reported for other salivary mucus (Lombart & Winzler, 1972;Roukema et al, 1976). Comparable data were unfortunately not provided for the RSM purified by Keryer et al (1973).…”

“…The N-acetylgalactosamine/(serine + threonine) ratio was 2.1: 1, which is compatible with the possibility that every serine and threonine residue may be linked to an oligosaccharide and that every oligosaccharide may contain an additional A-blood-group N-acetylgalactosamine residue as well as an N-acetylgalactosamine O-glycosidically linked to the peptide core. Galactose and sialic acid were also prominent, as is the case for mouse sublingual mucin (Roukema et al, 1976), but fucose was proportionately higher than in most salivary mucins isolated to date (Moschera & Pigman, 1975;Roukema et al, 1976) except canine submaxillary mucin (Lombart & Winzler, 1972). The sugar profile of our RSM, like the amino acid profile, bore little resemblance to that of RSM reported by Keryer et al (1973).…”

“…Tyrosine, phenylalanine, histidine, arginine and methionine were detected in low concentrations and totalled only 5.5% of all amino acids. RSM differs considerably from canine submaxillary mucin, which has an unusually high content of glycine (22.5%), and a threonine, serine and proline total of 34.5% (Lombart & Winzler, 1972).…”

“…blood-group-A determinant (terminal N-less well documented and, to our knowledge, the only publication on the isolation of RSM is that of Keryer et al (1973). However, the data provided by these authors are limited, and the carbohydrate and amino acid composition of their RSM is not consistent with those of other reported submandibular mucins (Roukema et al., 1976;Lombart & Winzler, 1972; Oemrawsingh & Roukema, 1974). The rat submandibular gland is rich in mucous acinar cells and has been extensively used, either in slice or dispersed-cell preparations, as an experimental model for studies on the mechanisms and control of mucus secretion (Bogart & Picarelli, 1978;Quissell & Redman, 1979;Quissell & Barzen, 1980;Fleming et al, 1980;Fleming & Sturgess, 1981).…”

Structure of mucous cells in salivary glands

“…The principal biochemical constituent of mucus is termed mucin. Mucin can be isolated from whole saliva (Veerman et al, 1989), from specific glands that have been ground (Lombart and Winzler, 1972;Oemrawsingh and Roukema, 1974), or from saliva obtained by means of a collecting device from a single gland or gland complex (Loomis et al, 1987). Biochemical analyses of mucin from various glands show that it consists mainly of carbohydrates (50-80%) and of protein composed of very long peptide chains festooned with numerous oligosaccharide side branches that range in length from two to ten sugar residues.…”