Biochemical and Molecular Biological Study on Sucrose Phosphate Synthase from Sweet Potato Suspension Cells Induced by Osmotic Stress

• Main Authors: Wei-Liang, Chen, 陳韋良
• Other Authors: Ping-Du Lee, PH.D.
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/60762149887099477308

博士 === 國立臺灣大學 === 農業化學研究所 === 91 === Sucrose phosphate synthase (SPS), a key enzyme in plant sucrose biosynthesis, is subject to the regulation of protein phosphorylation and allosteric effects. In order to elucidate the physiological role of SPS in sucrose futile cycle in sweet potato suspension cells, the following experiments were carried out. First, the variations of SPS activity in response to different concentration of carbohydrate in the presence of 0.6 M sorbitol were monitored. The cells harvested from medium without (normal treatment) or with (osmotic-stress treatment) 0.6 M sorbitol were compared. In those cells without treatment, the contents of sucrose, fructose and glucose were significantly lower than that of stressed-cells. Moreover, the activity of SPS in stressed-cells was 2~3 fold higher than normal cells. By applying cycloheximide, a cytoplasmic translation inhibitor, SPS synthesis was prevented and sucrose synthesis was lower in stress-cells. In the presence of okadiac acid, a potent protein phosphatase inhibitor, the SPS activity in stressed-cells shown two-fold higher than that from cells without okadiac acid. These observations indicated that SPS kinase and SPS activity are controlled at the level of gene expression and SPS also controlled by osmotic-induced phosphorylation. The second experiment was explore whether the osmotic-induced phosphorylation site and possible allosteric regulation site existed in the primary structure of SPS. To obtain the primary structure of SPS, the molecular cloning of SPS from sweet potato was carried out. The gene encoding SPS from tuber of sweet potato has been cloned, sequenced and expressed in Escherichia coli BL21 (DE3). The cDNA encoded for 1048 amino acids with predicted molecular mass of 116 kDa. The deduced amino acid sequences shown 66-80% identity with SPSs from spinach and potato, and possessed motifs for the phosphorylation site which may associate with light- and osmotic stress-induced regulation. The SPS gene was subcloned into pET-21b and subjected to heterologous expression in E. coli BL21 (DE3). Purification of recombinant SPS from E. coli employed His-tagged affinity column and FPLC Mono-Q anion-exchange chromatography. The purified protein was active and shown as a clear major band close to the expected molecular mass of 116 kDa. The heterologously expressed protein from E. coli was not allosterically regulated by G 6-P and Pi, and the Km for UDPG and F 6-P were 21.3 and 7.3 mM respectively. The recombinant proteins displayed a specific activity of 0.92 mmol per minute per mg protein, and an optium pH at 7.5. The enzyme was activated by Mg 2+, Ca2+ and Mn2+. The nucleotides, UDP, UTP inhibited the enzyme activity about 30-40%, and the sugar and sugar phosphates have little or no effect on its activity. Since the sweet potato SPS was not allosterically regulation by Glc6P and Pi, we speculate that cells cultured in osmotic-stress medium the SPS activity increase was not only regulated by gene expression but also affect by SPS-kinase. The possible mechanism of osmotic-stress to activate the SPS activity is as following: First, the SPS-kinase that does phosphorylation of SPS is on ser-416, secondary SPS increase gene expression level, both effects to make SPS activity raised in stress medium. Hence increase sucrose concentration in stressed cells. The metabolites of sucrose degradation and resynthesis may participate in starch synthesis and other pathway cycle to maintain cells growth in the stressed medium.