Multiple forms of α-galactosidase in mature leaves of Cucurbita pepo

Abstract

α- D-Galactosidase has been purified from mature leaves of Cucurbita pepo using pH and ammonium sulphate fractionation, Sephadex gel filtration and DEAE Sephadex gel chromatography. Gel filtration produced one peak of α-galactosidase activity from which three distinct enzyme forms were resolved on DEAE Sephadex and designated LI, LII and LIII. Purirications obtained were ca 75, 120 and 30 fold for LI, Lll and LIII respectively. Ll was slightly contaminated with β-galactosidase and LII with β-fructosidase activity. All forms hydrolysed the α-galactosyl linkages of raffinose and stachyose. Differences between each form were found in their pH optima, reactivity toward metal ions, thermal stability and K m values using either p-nitrophenyl-α- D-galactoside (NPG) or raffinose as substrates. All forms were inhibited by NPG at high concentrations and by α- D-galactose. It is proposed that α-galactosidases may be components of a lysosomal system in plant cells.