impact seriously on inhibitory activity but could potentially influence dimerization of the inhibitor, in which the carboxy- terminal domain has a suggested role. Mutant and wild-type segregants were selected from backcrosses and bulked BC2F3 and BC2F4 seeds of validated mutant and wild-type lines of the three families used for biochemical assays. Analysis of total seed protein and albumin profiles by protein gel electrophoresis and measurement of the amounts of these protein fractions did not reveal any significant difference between the mutant and wild-type lines within any one family. Measurement of overall trypsin and chymotrypsin inhibitory activities of seed protein extracts revealed a number of significant differences between mutant and wild-type lines. For the C77Y family, a significant reduction of greater than 60 was apparent for both trypsin and chymotrypsin inhibitory activity in mutant compared with wild-type lines. For the S85F family, a small but significant increase in TIA and a decrease in CIA were apparent in mutant compared with wild-type lines. For the E109K family, a slight but not significant decrease in TIA and CIA was apparent in mutant compared with wild-type lines. The same trends were observed for mutant compared with wild-type lines for TIA , CIA and when expressed on a seed meal or seed protein basis. The differences in inhibitory activity among mutants were investigated further following fractionation of the different isoforms corresponding to the PZ-51 closely related TI1 and TI2 genes in pea. The major isoforms in seeds have been shown to correspond to mature and carboxy-terminally processed forms for each gene product. Figs 3�C5 show the profiles of total protein extracts from mutant and wild-type families , when seed proteins are separated by GSK-1349572 structure cation-exchange chromatography and assayed for their ability to inhibit trypsin and chymotrypsin. Four isoforms were apparent among the separated seed proteins from all wild-type control lines ; the four isoforms were evident as fractionated protein peaks with the ability to inhibit both trypsin and chymotrypsin. Peptides from each of these four peaks were identified in seeds from wild-t
