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Metabolism and Enzymology

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves ¹

Seed Biosynthesis Research Unit, United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois, 61604

Galactinol synthase catalyzes the first committed step in the biosynthesis of raffinose sugars. Previous attempts to purify the enzyme have proven difficult and have resulted in low quantities of unpurified enzyme. Galactinol synthase was purified 752-fold from mature zucchini (Cucurbita pepo L. cv Burpee Hybrid) leaves using sequential liquid chromatography on DE 52, Octyl-Sepharose CL-4B, and Sephacryl S-200. This isolation scheme resulted in an 18.6% recovery of the initial activity. The purified enzyme had a specific activity of 23.3 micromoles per minute per milligram protein, a pH optimum of 7.5, and the activity was enhanced by dithiothreitol and MnCl₂. The enzyme was only half as active with MgCl₂ as with MnCl₂. Na⁺, K⁺, and Ca²⁺ cations had little effect on the enzyme activity, while Co²⁺, Zn²⁺, Cu²⁺, and Fe³⁺ cations were strongly inhibitory at 10 millimolar concentrations. Purified galactinol synthase bound specifically to the substrates myo-inositol and UDP-galactose (K_m = 6.5 and 1.8 millimolar, respectively), while exhibiting little affinity for an alternative glycosyl donor (UDP-glucose) or inositol epimers (epi- and scyllo-). Ten millimolar concentrations of UMP, UDP, UTP, AMP, ADP, ATP, NAD⁺, NADH, NADP⁺, UDP-xylose, and UDP-mannose, or 20 millimolar sucrose, talose, galactose, glucose, xylose, and melibiose exhibited various degrees of inhibitory effects. Twenty millimolar stachyose, raffinose, fructose, and mannose, and 10 millimolar UDP-glucuronic acid and UDP-galacturonic acid had little or no effect on the enzyme activity. The purified galactinal synthase is a monomer of M_r 42,000 with an isoelectric point of 4.1.

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