Purification and characterization of monkey (Macaca nemestrina) tracheobronchial mucin.
A major mucin glycoprotein was purified from monkey (Macaca nemestrina) bronchoalveolar lavages by gel filtration, delipidation, and a series of density gradient centrifugations in cesium trifluoroacetate/guanidinium chloride. Lipids noncovalently associated with the mucin amounted to 24-36% by weight and consisted primarily of phospholipids and glycolipids. The mucin preparation was free of low-molecular-weight protein/glycoprotein contaminants, glycosaminoglycans/proteoglycans, and nucleic acids. The weight-average molecular weight and radius of gyration of the mucin in buffer containing 6 M guanidinium chloride was estimated to be approximately 1.56 x 10(6) and 100 nm, respectively, by laser light scattering technique. When the mucin was dissolved in 0.15 M NaCl, a considerably higher molecular weight of approximately 5.05 x 10(6) and a larger radius of gyration of approximately 127 nm were observed suggesting aggregation of the mucin molecules. Amino acid composition of the glycoprotein was characteristic of mucins with threonine, serine, glutamic acid, proline, glycine, and alanine comprising 63%. The total carbohydrate content was 71.5% and consisted of GalNAc, GlcNAc, Gal, sialic acids, and fucose in the molar ratio of 1.0:2.2:2.4:1.4:1.2 with no detectable mannose. Alkaline borohydride treatment indicated that 65% of the threonine and 27% of the serine are substituted by saccharides via GalNAc residues. An antisera produced against the purified mucin was found to react well with the native and weakly with the deglycosylated mucins and will be useful for immunoassays. A second, minor, mucin glycoprotein obtained during the purification was also partially characterized.
Devaraj
H
H
Department of Biological Chemistry, M. S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Griffith
J W
JW
Sheykhnazari
M
M
Naziruddin
B
B
Sachdev
G P
GP
Bhavanandan
V P
VP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000032:analysis; D006031:Glycosylation; D008027:Light; D008055:Lipids / Q000032:analysis; D008254:Macaca nemestrina; D008970:Molecular Weight; D009077:Mucins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010743:Phospholipids / Q000032:analysis; D012542:Scattering, Radiation
D000596:Amino Acids; D002241:Carbohydrates; D006017:Glycolipids; D008055:Lipids; D009077:Mucins; D010743:Phospholipids