光照與滲透逆境下水稻蔗糖磷酯合成酶性質鑑定與免疫組織定位

Characterization and Immunolocalization Studies of Rice Sucrose Phosphate Synthase under Illumination and Osmotic Stress

李木和(Mu-Ho Lee)

指導教授 ： 李平篤

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.00877

摘要

以台農67號水稻蝴窱o芽所得白化苗、綠化苗及蝴篫a浮培養細胞，經光照、高滲透逆境 (0.6 M sorbitol) 處理為材料，在純化後以SDS-PAGE及native-PAGE配合抗体免疫分析，得知 蔗糖磷酯合成酶 (sucrose phosphate synthase, SPS) 次單元體分子量為116~120 kDa，原態分子量 420與520 kDa，推測 SPS可能以四元體的結構存在。
在生化性質方面：四種不同材料在生長週期中，SPS酵素在第三至七天活性較高，反應最適pH值均介於6~8之間，最適反應溫度為35~40℃，從白化苗與高滲透逆境處理之蝴篫a浮細胞分離出之SPS，對基質Fru 6-P及UDPG之Km值相近，約為1.8及35 mM；相對地，綠化苗與光照蝴篫a浮細胞SPS之 Km值，約為1.5及28 mM。在調控機制方面：白化苗與高滲透逆境處理之蝴篫a浮細胞之SPS，不會受到Glc 6-P的促進及Pi的抑制，然而綠化苗與光照蝴篫a浮細胞SPS則會受到Glc 6-P的促進及Pi之抑制。推論水稻中存在有二種型式SPS異構酶，分別命名為 SPS-I與SPS-II。SPS-I會受到滲透逆境的影響，SPS-II可受光的誘導。鎂離子對酵素活性有促進作用。核苷酸 (ATP, ADP, AMP, UTP, UDP, UMP)會降低SPS活性約40-50%。 硫氫化合物可抑制水稻 SPS 酵素活性，但加入DTT與

關鍵字

光照 ； 蔗糖磷酯合成酶滲透逆境

並列摘要

Sucrose phosphate synthases (SPSs) of rice (oryza sativa cv Tainong 67) were isolated and purified from etiolated rice seedlings (ERS), green rice seedlings (GRS), rice grain suspension cells under osmotic stress (RGSO) and rice grain suspension cells under illumination (RGSI). A native molecular mass of ca. 420 and 520 kDa were found using native-PAGE. The SDS-PAGE analyses revealed SPSs to be homotetramers composed of subunit with a mass of 116-120 kDa. The maximum activity for SPSs was observed on the third day after germination.
As far as their biochemical characterization was concerned, the optimum pH of the enzyme reactions lay generally between 6-8, the optimum temperatures between 35-40℃. The ERS and RGSO SPS Km values for Fru 6-P and UDPG were 1.8 and 35 mM, respectively. However, the GRS and RGSI SPS had a similar Km values for Fru 6-P and UDPG of 1.5 and 28 mM, respectively. GRS and RGSI SPS activities were allosterically regulated by Glc 6-P (activator) or Pi (inhibitor), but ERS and RGSO SPS had no effect. From their regulations and Km values two enzyme forms (SPS-I and SPS-II) could be discriminated in the rice. SPS-II was induced by illumination, but SPS-I by osmotic stress. All SPSs were activated by Mg2+. The nucleotides AMP, ADP, ATP, UMP, UDP and UTP inhibited enzyme activity by about 40-50%. Thiol reagents became sensitized to the enzyme activity, but could be restored with DTT or b-ME. The SPS activity were activated by glucose, galactose, glucosamine, maltose, and lactose but were inhibited by d-gluconolactone and mannose. SPSs were also inhibited by PCMBS, cibacron blue F3G-A and DEP.
Sucrose phosphate synthases were very unstable. The enzymes were easily to be degradated and deactivated in the purifying process. The experiment result points out, firstly, the SPS possibily breaked into two fragments at the position of NG for the KNGGP. It could cause the Asn and Asp deamidated easily, when dissolve in the strong acid, base, high temperature and high ionic strength. It made the enzyme protein to degradation. N-terminus of SPS contains UDPG, Fru 6-P binding site, NLC sequence and phosphorylation site; C-terminus contains SPP binding region, and SKL sequence. A histological observation revealed an abundance of starch granules in the light and osmotic stressed cells. SPS activity was determined in the surface of starch granules and cytoplasm. In summary, it was demonstrated that SPS activity and starch granules synthesis may participate for maintaining cell growth in the stress。

並列關鍵字

Sucrose phosphate synthase ； illumination ； osmotic stress

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