Human respiratory mucous glycoproteins.
Biochemical characterization of human respiratory mucus has generally utilized expectorated specimens. In order to exclude extraneous contaminants in the analysis of airway glycoproteins, human airways were cultured and the mucous glycoprotein released into the supernatant analyzed. By incorporating 3H-labeled glucosamine or 14C-threonine into the media, the airways biosynthetically labeled the mucous glycoproteins (MGP), facilitating their analysis. The MGP chromatograph by gel filtration on Sepharose 2B in two fractions: one excluded from the column and one that enters the column. However, employing a gel filtration column with the ability to fractionate larger molecules, Sephacryl S-1000, it was found that MGP fractionate over a large range in molecular sizes and do not segregate into distinct fractions. The diffuse, broad peak of MGP fractionation on Sephacryl S-1000 is not affected by reduction and alkylation or by chromatography in 1 M NaCl. The fractionated MGP from Sepharose 2B were divided into larger and smaller molecular species, and their charge characteristics were determined by DEAE chromatography and preparative isoelectric focussing. MGP exhibit strong acidic charge characteristics that are uniform, as reflected in elution from DEAE and a single, sharp isoelectric focussing point. Enzymatic cleavage of the oligosaccharide side chains from MGP liberates more than 70% of the radiolabeled side chains. The side chains enzymatically cleaved from the larger and smaller molecular species of MGP are similar in size. Highly purified MGP were found to be 73% carbohydrate and 27% protein. Thus, human airways release a family of MGP that express marked heterogeneity in size but a uniform, strong acid charge and include side chains of similar size.
D050260:Carbohydrate Metabolism; D005591:Chemical Fractionation; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002849:Chromatography, Gas; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009093:Mucus / Q000378:metabolism*; D009924:Organ Culture Techniques; D012137:Respiratory System / Q000378:metabolism*; D000519:alpha-Galactosidase / Q000494:pharmacology
D006023:Glycoproteins; C039044:N-acetyl-alpha-D-galactosaminyloligosaccharidase; D000519:alpha-Galactosidase