Stachyose synthesis in leaves of Cucurbita pepo
An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC126.96.36.199), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (Km 7.7 mM) and the acceptor raffinose (Km 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co2+, Hg2+, Mn2+ and Ni2+ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (Ki 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.