Fig. 1. Preparation of Muclin-depleted ZGC and in vitro aggregation of ZGC and purified Muclin. (A) Isolated soluble zymogens were depleted of Muclin on a PNA-agarose column. ZGC, starting material; FT, flow-through fractions; W, wash fractions; E, 0.2 M lactose eluted fractions. FT-4 and W-1 were pooled and used for the in vitro aggregation assay. (B) Muclin-depleted ZGC were incubated at pH 8.0 (Acid -) or acidified to pH 6.3 (Acid +). The aggregates were pelleted, run on 10% SDS-PAGE and stained with Coomassie blue. The control is 50 µg ZGC alone; `Muclin' included 5 µg purified Muclin, which increased ZGC aggregation compared with the control; `BSA' included 10 µg bovine serum albumin, which did not aggregate at acidic pH; `EGTA' included 5 mM EGTA to chelate calcium, which did not affect aggregation; `Ca²⁺' included 2 mM CaCl₂, which did not affect acid-induced aggregation. (C) Quantification of amylase in the pellets by densitometry (arbitrary units) of Coomassie-blue-stained gel. (D) Western blot for Muclin in the pellets. No Muclin was detected in the ZGC-depleted samples, but was detected only when purified Muclin was added (Muclin). The amount of Muclin in the pellet was increased upon acidification (Acid +). (E) By western blot, Muclin in the absence of ZGC does not exhibit acid-mediated aggregation.