Sucrose phosphate, preparation

Results obtained by Leloir and Cardini indicated that two separate enzymes are involved in the biosynthesis, in plants, of sucrose and sucrose phosphate from D-fructose and D-fructose 6-phosphate, respectively in the presence of UDP-D-glucose these enzymes have been partially separated. Slabnik and coworkers succeeded in isolating sucrose synthetase and sucrose 6-phosphate synthetase from potato tubers, and determined some of the properties of the partially purified preparations. The sucrose synthetase showed an optimum activity at 45 and was inhibited completely by ADP and some phenolic D-glucosides, whereas these had no effect on sucrose 6-phosphate synthetase. 

The evidence for the double function of the enzyme was obtained from the fact that when P -labeled inorganic phosphate and nonradioactive a-D-glucose 1-phosphate are added to sucrose phosphorylase preparations... 

The crude enzyme preparations contained a specific phosphatase for the sucrose phosphate and with these preparations sucrose was formed. 

Eleven-day-old dark-grown maize plants were used. Sixty-seven grams of leaf tissue was used for each preparation. Each set was permitted to absorb 0.4 mC T-phosphate. Preparation and sucrose density gradient centrifugation were as described for Twice washed 1000 g pellet, Fig. 6. 

Phosphate Esters. The phosphorylation of sucrose using sodium metaphosphate has been reported (78). Lyoptulization of a sodium metaphosphate solution of sucrose at pH 5 for 20 hours followed by storage at 80°C for five days produced a mixture of sucrose monophosphates. These products were isolated by preparative hplc, with a calculated yield of 27% based on all organic phosphate as sucrose monoesters. Small proportions of glucose and fmctose were also formed. 

Synthesis of sucrose 6 -phosphate by an enzymic method using uridine 5 -(a-D-glucopyranosyl diphosphate) plus D-glucose 6-phosphate has been reported.126-131 The first, unambiguous, chemical synthesis of sucrose 6 -phosphate was achieved by Buchanan and coworkers.18 The reaction of 2,3,4,6,l, 3, 4 -hepta-0-acetylsucrose, prepared by five steps of synthesis, with cyanoethyl phosphate in pyridine gave a crude product from which sucrose 6 -phosphate was isolated as the barium salt. 

The tissue blocks were immersed in 0.1 M phosphate buffer containing 2% glutaraldehyde and 5% sucrose (pH 7.4). The tissue blocks were immersed in ZIO solution. The ZIO solution was prepared as follows 3 g of zinc (powder) was dissolved ultrasonically in 20 mL distilled water and 1 g of iodine crystals was slowly added to the zinc suspension with stirring. After cooling, 4 mL of the filtered solution was mixed with 2 mL of Tris-HCl buffer (pH 7.5) and 2 mL of 2% Os04 solution. Treatment with this ZIO mixture was carried out for 16-20 h at 4°C in the dark. 

The direct syntheses of D-glucosyl-L-sorboside from sucrose and L-sorbose and of sucrose from D-glucosyl-D-xyloketoside and D-fructose can be obtained in phosphate-free enzyme preparations.40 The reactions are ... 

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