Abstract
The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].