Chromatographic measurement of mucin production in cultures of gastric mucous cells.
The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent.
Keogh
J P
JP
Department PF/GM, F. Hoffmann LaRoche Ltd., Basel, Switzerland.
Edelmann
A
A
Fischli
A
A
D000818:Animals; D002478:Cells, Cultured; D002847:Chromatography, Agarose / Q000379:methods*; D003513:Cycloheximide / Q000494:pharmacology; D005752:Gastric Mucins / Q000032:analysis / Q000096:biosynthesis*; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008970:Molecular Weight
D005752:Gastric Mucins; D003513:Cycloheximide