Partial purification and characterization of stachyose syynthase in leaves of Cucumis sativus and Cucumis melo: utilization of a rapid assay for myo-inositol

A rapid enzymatic assay to quantitate myo-inositol using myo-inositol dehydrogenase-linked NAD reduction has been utilized to measure stachyose synthase activity Stachyose synthase activity in extracts of source leaves from C. sativus as well as sweet (cv. Noy Yizre'el) and non-sweet (cv. Birdsnest) genotypes of C. melo was separated completely from galactosidase and galactohydrolase activity via MONO Q anion exchange chromatography. As a result, it was determined for the first time that galactohydrolase activity present in leaf extracts is inhibited in the presence of raffinose and does not interfere with accurate estimation of stachyose synthase activity. The apparent K_m values for stachyose synthase for galactinol were determined to be 4.6 mM, 2.4 mM and 3.0 mM for C. sativus, C. melo cv. Birdsnest and cv. Noy Yizer'el, respectively. The apparent K_m value for raffinose was estimated to be approximately 15 mM for both the cucumber and melon source tissues. No in vitro metabolite effectors of the partially purified stachyose synthase have been identified. Thus, in vitro regulatory and kinetic properties of source leaf stachyose synthase are similar between species or cultivars of Cucumis which are predicted to differ significantly in sink carbohydrate metabolism.