Studies on pig and sheep salivary mucins

Abstract

Sheep and pig submaxillary mucous glycoproteins have routinely been purified by selective precipitation (e.g. with cetavalon or acid methanol) followed by absorption (DEAE-cellulose or hydroxylapatite) (Gottschalk et al., 1972). These glycoproteins on sedimentation analysis are polydisperse and of high M, ranging from 500000 to 2.5 x lo6. Both glycoproteins have been reported to consist of polymers of subunits. In sheep submaxillary mucus (OSM) the subunits joined together non-covalently are dissociated by ionic interactions (Hill et al., 1977). In pig submaxillary mucus (PSM) the subunits are reported to be joined together by interchain disulphide links (Holden et al., 1971). Here we describe studies on the dissociation of OSM and PSM purified by equilibrium centrifugation in a CsCl density gradient, a method used for other gastrointestinal mucous glycoproteins (Starkey et al., 1974). Submaxillary mucus was extracted from fresh glands by homogenization in 0.2~-NaCl, 0.02% sodium azide (pH6.5-7.0) at 4°C. After equilibrium centrifugation in a CsCl density gradient the protein (fractions 1 and 2) separated from the glycoprotein, fraction 6 for OSM and 7 for PSM, with the nucleic acid in fraction 9. The content of sialic acid and galactosamine was 31% and 25% respectively for OSM and 15% and 23% respectively for PSM. These values agree with those for other purified preparations of submaxillary gland mucous glycoproteins (Carlson, 1968; Hill et al., 1977). On SDS/polyacrylamidegel no protein bands were visible either before or after reduction when stained with Coomassie blue and only traces of protein were detectable with silver nitrate. N-Terminal analysis showed the absence of any detectable Nterminal amino acids, except following proteolysis with papain when mainly alanine was detected for OSM and serine for PSM. The N-terminal amino acid of OSM has previously been shown to be blocked (Hill et al., 1977). These results show the submaxillary gland glycoprotein preparations to be free of all but insignificant amounts of noncovalent bound protein. From amino acid analysis the protein content was 39% for PSM and 52% for OSM. After gel filtration on Sepharose 2B, PSM was completely excluded and remained so after treatment with 8~-urea, 1% SDS or 3.5w-Cscl. Treatment of these glycoprotein preparations for 5h at pH2.0 (in the presence of a mixture of protease inhibitors), boiling for lOmin or 24h in 0.2~-mercaptoethanol dissociated the PSM into smaller sized glycoprotein components that were well included on Sepharose 2B. To demonstrate complete dissociation of PSM at pH 2.0 or by O.2w-mercaptoethanol it was necessary to run the column at an acidic pH or in the reducing buffer. Raising of the pH to 7.0 or removal of the 0.2~-mercaptoethanol resulted in reassociation of the glycoprotein into a component that was again excluded on Sepharose 2B and which was noticeably more viscous than the original undissociated glycoprotein. These results show that PSM is dissociated by non-covalent-bond-breaking agents or by breaking the disulphide links. It would appear therefore