Purification and characterization of cytosolic and cell wall β-galactosidases from Vigna unguiculata stems

Fabrício Bonfim Sudério, Gislainy Karla da Costa Barbosa, Enéas Gomes-Filho, Joaquim Enéas-Filho
2011 Brazilian Journal of Plant Physiology  
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
more » ... ich 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.
doi:10.1590/s1677-04202011000100003 fatcat:sqn4l6lffjbd7mdsppfndhrr7m