Figure 11.

Lack of an association between HD-5 and chymotrypsin/trypsin in crypt cell extracts. A: AU-PAGE Westerns of crypt epithelial extract and ileostomy fluid concentrate from same patient. Ileal crypt epithelial acid extract obtained from resection specimen of patient CD6 (lanes 1, 3, and 5) was separated by AU-PAGE in same gels as concentrated ileostomy fluid from the same patient (obtained 5 weeks after operation; lanes 2, 4, and 6). After transfer to PVDF membranes, immunoblotting was performed using antibodies to chymotrypsin (i), HD-5 (ii), and trypsin (iii). Chymotrypsin- and trypsin-immunoreactive bands in ileostomy fluid (lanes 2 and 6, respectively) have similar electrophoretic mobility as two HD-5 immunoreactive bands (dashed arrows, lane 4). By contrast, for ileal crypt epithelial cell extracts, electrophoretic mobility profiles of immunoreactive bands for chymotrypsin (lane 1), HD-5 (lane 3), and trypsin (lane 5) do not show any similarity. For HD-5 in ileostomy fluid (lane 4), the findings are consistent with the presence of the peptide in free mature form (solid arrow), and in a complex with trypsin and chymotrypsin (dashed arrows). B: Absence of chymotrypsin and trypsin in preparations of pro-HD-5 purified from small intestinal crypt extracts. RP-HPLC-purified pro-HD-5 from acid extracts of normal small intestinal crypt epithelial cells (lane 1) and acid extracts of crypt epithelial cells from two separate Crohn’s small intestinal resection specimens (lanes 2 and 3) were studied by dot blot analysis. Purified pro-HD-5 preparations from all three crypt extracts were immunoreactive for HD-5, but not trypsin or chymotrypsin.