Figure 6.

Fractionation of DBEs from nonmutant and su1-Ref kernels by gel-permeation chromatography. A, DBEs in nonmutant kernels. Proteins from nonmutant kernels harvested 20 DAP were applied to a Sephacryl S-200 superfine gel-permeation column. Fractions were assayed for pullulanase-type DBE activity by measuring formation of new reducing ends after incubation with pullulan (activity units, ▪), and for isoamylase-type DBE activity by determination of iodine-complex absorbance maxima after incubation with amylopectin (A₅₅₀, □). Equivalent amounts of protein from fractions exhibiting DBE activity were analyzed for the presence of ZPU1 or SU1 on immunoblots with the indicated antisera. B, DBEs in su1-Ref kernels. Proteins from su1-Ref kernels harvested 20 DAP were fractionated and assayed for DBE activity, and immunoblot analysis was performed, as described for A. C, Comparative immunoblot analysis. Nonmutant proteins in fractions 9 to 12 from A (lanes +) and su1-Ref proteins in fractions 9 to 12 from B (lanes m) were subjected to immunoblot analysis with the anti-ZPU1 antibody. Equivalent amounts of protein were loaded, and each lane contained twice as much protein as the immunoblots shown in A and B.