Galactosidase de cotilédones de Vigna unguiculata (L.) Walp. cv pitiúba: efeito da salinidade na atividade durante a germinação e estabelecimento da plântula, purificação parcial e propriedades.

Abstract

Pitiuba cowpea (Vigna unguiculata (L.) Walp.) seeds were sown in distilled water (controI treatment) and in 0.1 M NaCl solutions (saline treatment). The effects of salinity on α- and β-ga!actosidase activities were studied both in vivo and in vitro. The in vivo experiments were perfomed in both treatment by assaying these enzymes in cotyledon crude extracts obtained from seeds or seedlings along germination and seedling establishment, and the in vitro ones were perfomed by assaying the enzymes in cotiledon crude extracts from quiecents seeds as a function of increasing NaCI molarities. The activity of both enzymes was present at higher levels in the quiescent seed. α-galactosidase activity increased during the first day after sowing, stayed constant up to the 3rd day, and then started to decrease up to the end of the experimental period. β-galactosidase activity showed the same trend, however, it started to drop only after the 5th day. The addition of 0.1 M NaCl to the germination medium (in vivo experiments) retarded the development of the activity of both enzymes, however, when the same concentration of this salt was added in vitro it had no effect on the activities of both enzymes. The obtained results suggest that salinity delays galactosidases and/or affected their turnover throughout germination and seedling establishment. In order to purify α-galactcsidase a cotyledonary crude extract obtained from quiescent seeds was precipitated with citric acid to pH 3.5 and the supernatant was fractionated by ammonium sulphate between 20-85% saturation, and gel-filtered through a Sephadex G-100 column. Using this procedure it was obtained one peak of activity which gave an enzyme purification of 9.0 fold and an activity vield of 29.0%. When this fraction was subjected to fractionation on a CM-Cellulose column only one peak of α-galactosidase was obtained. However, this additional step gave an enzyme purification of only 3.5 fold and an activity yield of 2.4%. The molecular weight of the partially purified enzyme determined by gel-filtration was 43.500, and it was observed onIy one cotyledonary α-galactosidase with the same molecular weight at the 5th day after sowning throughout germination and seedling establishment. Salinity did not caused the appearance of new forms of α-galactosidase. This partially purified enzyme had an optimal pH of 6.0 and it was strongly inhibited by galactose and Hg2+, moderately inhibited bv Fe2+, Mn2+, Co2+, Cu2+ and EDTA, but Ca2+, Mg2+ as well as the reducíng agent 2-mercaptoethanol had no effect on its activity. Its thermal stability was similar to other plant α-galactosidases.