The separation and characterization of bronchial glycoproteins by density-gradient methods

Abstract

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.

Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma.

“…For electrophoretic and immunoblot analysis, DU-PAN-2 antigen was also purified by cesium bromide density gradient centrifugation (6) V, overnight in the cold. The sheet was then incubated with 5% bovine serum albumin at 40°C followed by DU-PAN-2 antibody or a control antibody at room temperature for 1 hr with continuous rocking.…”

Isolation and partial characterization of rat gastric mucous glycoprotein

“…For this reason they can be almost completely separated from non-cova]ently bound protein by equflibrium centrifugation in density gradients of highly concentrated cesium salt solutions. This method was used successfully to isolate mucous glycoproteins from pig gastric mucosa [1], from human ovarian cyst fluids [7], from sputum [8] and from cervical mucus [9]. In the experiments described in this paper gastric mucous glycoproteins from rat were isolated on cesium chloride gradients.…”

Biochemical Properties of Tracheobronchial Mucins from Cystic Fibrosis and Non-Cystic Fibrosis Individuals

“…Since proteinases in the C F LM-gel samples fragmented radiolabeled tracheobronchial mucin In vitro (Fig. 4), proteinases in LM-gel samples from pathological airways may account for the variations in molecular weight, solubility, and chromatographic behavior reported for tracheobronchial mucins (6,(37)(38)(39)(40)(41)(42)(43)46). This study has also demonstrated that TBM from healthy individuals exhibited physical and chemical properties similar to those of TBM from a patient with bronchial asthma (5).…”

“…4) The cysteine contents of the CF lung mucins analyzed were considerably lower than those of normal and asthmatic mucins, and similar to values obtained on analysis of the proteolytically-digested glycopeptide fragments of asthmatic mucin (39,40). However, considerable variability in the cysteine concentrations of tracheobronchial mucins has been reported (5,6,33,(38)(39)(40)(41)(42)(43) and cysteine is often not measured quantitively as it requires almost a mg of purified mucin for accurate measurements in duplicate.…”

“…While the V, peak was referred to as mucin, no characterization or analyses were reported to substantiate this claim. On the other hand, the mucin sample isolated from bronchial washings of healthy airways by Sachdev et al (12) has a molecular weight of 69,400 daltons which is markedly smaller than the molecular weight values of 2 to 7 x lo6 obtained with asthmatic, bronchitic and/or bronchorrhea TBM (38,42,46) as well as with TBM isolated from tracheal aspirates of nonpulmonary patients (33,43). The normal mucin isolated by Sachdev et a1 (12) is also smaller than the tracheobronchial glycopeptide fragment (MW = 150,000) obtained on pronase digestion of BA mucin (38).…”