A simplified electrophoretic assay for human salivary α-amylase inhibitor detection in cereal seed flours

Alpha-amylase inhibitors are antinutritional proteins largely found in cereal seeds. An in-gel assay was developed that allowed the rapid screening of these compounds in complex seed extracts. The assay was based on the electrophoretic separation of the extract proteins on starch-containing gels, followed by the detection of alpha-amylase-inhibiting proteins after incubation of the gel in an alpha-amylase solution; inhibitors were revealed by a staining method based on iodine binding to nondigested starch. The one-dimensional method can be useful to test inhibitory activity of purified proteins or to assay fractions recovered during a purification procedure. A two-dimensional (IEF x PAGE) non-denaturing system with second-dimension separation on starch-PAGE was also developed; the technique allowed the screening of complex protein mixtures for multiple inhibitory proteins. The newly developed assay method was used to test the presence of inhibitory activity in a crude extract from wheat flour, and it was validated by comparing in-gel and in-solution assays of commercially available alpha-amylase inhibitors

Major proteinases hydrolyzing gliadin during wheat germination

A proteinase, representing the bulk of the enzyme activity for the hydrolysis of gliadin, was extracted from endosperms isolated from germinated seeds (four days) and was purified by ion-exchange chromatography and preparative isoelectric focusing. The optimal pH for gliadin hydrolysis was 4.25. The M r, determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 30 000; the isoelectric point was 4.5. The enzyme activity was totally inhibited by E-64 and cystatin, while inhibitors of other classes of proteinases were barely effective or ineffective. The activity was stimulated by sulphhydryl compounds. The proteinase hydrolysed to small peptides the gliadins from durum and soft wheat seeds. Other protein substrates were weakly degraded or not degraded. The proteinase appears to belong to the cysteine class and to play a key role in the initial mobilization of the main reserve protein in the starchy endosperm

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death