-Three isoforms of β-galactosidases were isolated and partially purified from the cotyledons of quiescent seeds of Vita 3 and Vita 5 cowpea [Vigna unguiculata (L.) Walp.] cultivars differing in water and salt stress tolerance. The purification procedure consisted of ammonium sulfate fractionation, acid precipitation, ion exchange chromatography through DEAE-sephadex and affinity chromatography through Lactosyl-sepharose columns. The three isoforms isolated from the two cultivars showed the same chromatographic patterns, same optimum of temperature for enzyme activity assay (60ºC), identical thermal stability up to 50°C, and similar pH optima (3-4). However, they differed from each other in sensitivity towards metal ions and certain chemical agents presents in the assay medium. The results have shown that the observed differences in β-galactosidases from the cotyledons of quiescent seeds were not sufficient to relate them to stress tolerance. ADDITIONAL INDEX TERMS:cotyledons, isozymes, Vigna unguiculata, Vita 3 and Vita 5 cultivars, salt tolerance. ISOLAMENTO E PURIFICAÇÃO PARCIAL DE β-GALACTOSIDASES DE COTILÉDONES DE DOIS CULTIVARES DE FEIJÃO-DE-CORDARESUMO -Três isoformas da enzima β-galactosidase foram isoladas e purificadas parcialmente a partir de cotilédones de sementes quiescentes dos cultivares Vita 3 e Vita 5 de feijão-de-corda [Vigna unguiculata (L.) Walp.], os quais diferem em tolerância aos estresses hídrico e salino. O processo de purificação consistiu de fracionamento com sulfato de amônio, precipitação ácida, seguida pela associação de cromatografia de troca iônica em coluna de DEAE-Sephadex com cromatografia de afinidade em coluna de Lactosyl-Sepharose. As três isoformas isoladas dos dois cultivares mostraram os mesmos padrões cromatográficos, mesma temperatura ótima de ensaio para atividade enzimática (60ºC), idêntica estabilidade térmica até 50ºC e apresentaram ótimos de pH semelhantes (3-4). Entretanto, diferiram umas das outras em suas sensibilidades a íons metálicos e a certos agentes químicos presentes no meio de reação. Os resultados mostraram que as diferenças observadas nas β-galactosidases de cotilédones de sementes quiescentes foram insuficientes para correlacioná-las com a tolerância ao estresse.

Three β-galactosidase isoforms, β-gal I and β-gal II (cytosolic) and β-gal III (cell wall-associated), were isolated from stems of Vigna unguiculata (L.) Walp. cv. Pitiúba seedlings. Purification consisted of ammonia sulfate fractionation followed by chromatography in DEAE-Sephadex and Lactosyl-Sepharose columns. The two cytosolic isoforms showed the same chromatography pattern, which differed from that of β-gal III. Electrophoresis revealed a single band of protein for β-gal II and β-gal III which also expressed β-galactosidase activity in gel. The apparent molecular mass of the β-gal I, II and III was 89, 146 and 124 kDa, respectively. The three isoforms revealed the same optimal pH (4.0) and the same optimal assay temperature (55°C) for enzyme activity. The three isoforms were stable at temperatures up to 50°C, and incubation with glucose and galactose expanded their thermal stability as well as inhibited their activities. Galactose was the most effective in promoting these effects and β-gal I and II were competitively inhibited by this sugar. Kinetic analysis using β-PNPG as substrate, revealed KM of 1.69, 1.76 and 1.43 for β-gal I, β-gal II and β-gal III, respectively. The β-gal I was able to hydrolyze all synthetic substrates tested, whereas β-gal II exhibited only β-fucosidase and a-arabinosidase activities, and β-gal III was limited to the a-galactosidase, β-fucosidase and a-arabinosidase activities. These results are consistent with three distinct β-galactosidases exhibiting quite similar kinetic features, but endowed with different functional specificities probably related to their specific roles in the plant cell physiology.

ABSTRACT(Salt stress delays morphophysiological development and activation of cell wall galactosidases in Vigna unguiculata stems). In order to examine the participation of α-and β-galactosidases in the cell expansion of stems from cowpea seedlings submitted to salt stress during plant establishment as well as to analyze the eff ect of salt stress on the development of seedlings and enzymatic activities, Pitiúba cowpea seeds were sown in distilled water and in 100 mM NaCl. Th roughout seed germination and seedling development, stems were harvested from seedlings at diff erent stages of development and at diff erent times aft er planting. Growth was evaluated by measuring stem length and fresh and dry mass of stems. Salinity both inhibited and delayed the growth of seedling stems. Th e eff ects of NaCl on galactosidase activities of the cell wall were studied, both in vivo and in vitro. Galactosidase activities in vivo were related to eff ects of NaCl inhibition and delay of stem development. Th e increase in salt concentration inhibited isolated galactosidase activity of cell wall from stems of seedlings. Starting at 250 mM NaCl, β-galactosidases were more sensitive to salt than α-galactosidases.