Proteins were extracted from untransformed and transformed
tobacco seeds as previously described (Grossi de
Sa and Chrispeels, 1997) by grinding 250 mg of dry tobacco
seeds, in an ice-cold mortar with 1 mL of 50 mM Tris pH
8.0 containing 30 mM NaCL, 0.1% Triton X-100 and 2%
2014 R. de Azevedo Pereira et al. / Phytochemistry 67 (2006) 2009–2016
b-mercaptoethanol. For inhibitory enzyme assays, proteins
were extracted in the same buffer in the absence of bmercaptoethanol.
Extraction was carried out for 2 h at
4 C with agitation. The extract was cleared by centrifugation
at 12,000g for 10 min and the supernatant was used as
a source of inhibitor for a-amylase assays. Protein concentration
was measured by the Bradford (1976) procedure.
Bovine serum albumin was used as standard.