Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Abstract

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 MnCI2. The enzyme was only half as active with MgCI2 as with MnCI2. 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 myoinositol and UDP-galactose (Km = 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 Mr 42,000 with an isoelectric point of 4.1.The raffinose family ofoligosaccharides is the second largest group of sugars present in plant tissues (5). In many species, particularly the Cucurbita, these oligosaccharides (raffinose, stachyose, and verbascose) serve as transport sugars (19). Raffinose saccharide biosynthesis is initiated by the transfer of a galactosyl unit from galactinol (O-a-D-galactopyranosyl myo-inositol) to Suc to produce raffinose; this reaction is further iterated to yield stachyose and verbascose (15). Galactinol is synthesized from UDP-Gal and myo-inositol by UDP-D-a-galactose:inositol galactosyltransferase or GS2 (15) as shown below:UDP-Gal + myo-inositol *-* galactinol + UDP 'Supported in part by a grant from the American Soybean Association to T. M. K.

Soybean seed galactinol synthase activity as determined by a novel colorimetric assay

“…The pH and temperature profiles obtained for the partially purified enzyme match exactly that obtained with the enzyme sample resulting from the treatment of the seed extract with MnCl 2 (Figure 4A,B). The optimal pH value is comparable to that reported for the GS enzyme from kidney bean cotyledons, which had an optimal pH of 7.0 (Liu et al, 1995), and to that reported for the enzyme purified from zucchini squash leaves, which had a broad pH optima from 7.0 to 8.0, with activity dramatically decreasing below 6.5 and above 8.0 (Smith et al, 1991). The activity of the partially purified GS enzyme was enhanced by MnCl 2 ( Figure 5) and DTT (not shown).…”

“…Although GS has been purified from Cucurbita pepo (Webb, 1982;Smith et al, 1991;Liu et al, 1995) and kidney bean cotyledons (Liu et al, 1995), purification of soybean seed GS has not been reported yet. A partial purification protocol for soybean seed GS and some of the its kinetic properties are therefore reported here.…”

Water Deficit Effects on Raffinose Family Oligosaccharide Metabolism in Coleus

“…GS activity was determined as previously described (Smith et al, 1991) with a few modifications. The reaction was carried out in a microfuge tube in a final volume of 100 L, with the following concentrations of substrates: 20 mm myoinositol, 3 mm DTT, 10 mm MnCl 2 , 2 mm UDP-Gal containing 30,000 dpm UDP-[ 14 C]Gal (specific activity ϭ 59 mCi mmol Ϫ1 ; ICN, Costa Mesa, CA).…”

Identification and Functional Characterization of the GALACTINOL SYNTHASE (MoGolS1) Gene in Melissa officinalis Plants

“…Because the only known function of galactinol is the formation of RFOs [Saravitz et al, 1987;Liu et al, 1995], which is putatively the committed enzyme step in the RFO biosynthetic pathway [Pridham and Hassid, 1965;Saravitz et al, 1987;Smith et al, 1991;Peterbauer and Richter, 2001] from UDPgalactose. Thus GolS catalyses the first step in the biosynthesis of galactinol and RFO from UDP-galactose and plays regulatory roles in carbon partitioning between sucrose and RFOs [Saravitz et al, 1987] under the abiotic stress conditions, and that galactinol and rafinose function as osmoprotectants in droughtstress tolerance of plants.…”