Macromolecular properties and polymeric structure of canine tracheal mucins.
Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.
Shankar
V
V
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Virmani
A K
AK
Naziruddin
B
B
Sachdev
G P
GP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel / Q000379:methods; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008027:Light; D046911:Macromolecular Substances; D008970:Molecular Weight; D009077:Mucins / Q000737:chemistry* / Q000302:isolation & purification; D009092:Mucous Membrane / Q000378:metabolism; D009439:Neuraminidase; D012542:Scattering, Radiation; D014132:Trachea / Q000378:metabolism*
D000596:Amino Acids; D002241:Carbohydrates; D046911:Macromolecular Substances; D009077:Mucins; D009439:Neuraminidase