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題名:Purification and characterization of a cytosolic starch phosphorylase from etiolated rice seedlings     (20點)
水稻白化苗澱粉磷解?之純化與生化性質研究
著者:Jen-Hung Hsu(許仁弘) ;Chien-Chih Yang(楊健志) ;Jong-Ching Su(蘇仲卿) ;Ping-Du Lee(李平篤)
出版地區:台灣
出版城市:台北市
學科:生命科學
關鍵字:澱粉磷解? ; 水稻 ; 葡聚醣 ; 糊精 ; 親和性層析 ; Affinity chromatography ; Dextrin ; Glucan ; Rice ; Starch phosphorylase
刊名:Botanical Bulletin of Academia Sinica
頁碼:187-196
語言:英語
摘要: 中文摘要PDF ; 英文摘要PDF

澱粉磷解?(starch phosphorylase, 簡稱SP)為參與植物澱粉代謝的重要酵素之一,其催化由葡萄糖-1-磷酯(Glc 1-p) 合成α-1,4-葡聚糖(a-1,4-glucan)之可逆反應。在植物中,普遍具有H與L型SP。但是在水稻白化苗中,我們發現水稻澱粉磷解?(rice starch phosphorylase, RSP)以H型為主要型式,而L型則極少,目前已成功純化H-RSP蛋白質。以台農67號水稻白化苗為材料,經硫酸銨分劃、陰離子交換層析(DEAE-Sepharose CL-6B)、親和層析(dextrin-Sepharose 4B),可得到均質H-RSP。純化倍數約300倍,回收率約21%。經FPLC Superose 12膠體過濾鑑定,其原態分子量為145kDa。由SDS-PAGE電泳,得知其次單元體分子量為 85kDa。最適反應pH值為5;最適反應溫度為45~50℃。在合成方向上,H-RSP對於Glc 1-P之Km值為 2.1mM;Vmax值為 5.85 Umg-1。在磷解方向上,對磷酸根之Km值為 3.8 mM。並且對於支鏈澱粉、肝醣、可溶性澱粉及糊精之親和性高,而對於麥芽寡糖(malto-oligosaccharide, G6~10)親和性低。另外,H-RSP對於糊精之Km值,為支鏈澱粉的9倍。環糊精六醣、環糊精七醣、環糊精八醣與麥芽四糖,為H-RSP抑制劑。而Mannose 1-P、Fru 6-P、ADPGlc、UDPGlc、AMP、IMP與PEP也會抑制酵素活性。金屬離子Ag+、Hg2+、Zn2+會降低酵素活性。但硫醇基修飾化合物卻可促進酵素活性,顯示其可能與酵素穩定性之維持有關。
Starch phosphorylasel (Pho1) from etiolated rice (Oryza sativa L. cv. Tainong 67) seedlings was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B anion exchange chromatography, and dextrin-Sepharose 4B affinity chromatography. The purification fold was 299, and the enzyme activity recovery was about 21%. The molecular mass of the native Pho1 on Superose 12 gel filtration was 145 kDa. The subunit molecular weight as determined by SDS-PAGE was 85 kDa. The enzyme has an optimum pH of 5 and an optimum reaction temperature of about 45℃~50℃. In the synthetic reaction for Glc 1-P, the Km value was 2.1 mM, and the Vmax value was 5.85 U mg -1. In the phosphorolytic direction for orthophosphate, the Km value was 3.8 mM. Pho1 has a higher affinity for amylopectin, glycogen, soluble starch and dextrin than for maltooligosaccharide (6 to 10 glucose units). In addition, the Km value for amylopectin was ninefold lower than for dextrin. Cyclohexaamylose, cycloheptaamylose, cyclooctaamylose, and maltotetrose were inhibitors of Pho1. Mannose 1-P, Fru 6-P, ADPGlc, UDPGlc, AMP, IMP and PEP also inhibit Pho1. The metal ions Ag+, Hg2+, and Zn2+ also reduce the enzyme activity. However, thiol reagents activate Pho1 activity, suggesting that sulfhydryl-group(s) may be required for enzyme stability.


    

本卷期目次
Botanical Bulletin of Academia Sinica
Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') constituents/ Dong-Jiann HuangChun-Der LinHsien-Jung ChenYaw-Huei Lin
Purification and characterization of a cytosolic starch phosphorylase from etiolated rice seedlings/ Jen-Hung HsuChien-Chih YangJong-Ching SuPing-Du Lee
Changes in soluble sugar content and respiration rate in methyl jasmonate-treated rice leaves/ Chien Teh ChenYen-Shuo SuChing-Huei Kao
Copper-induced stress and antioxidative responses in roots of Brassica juncea L./ Song-Hua WangZhi-Min YangHong YangBo LuShao-Qong LiYa-Ping Lu
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Comparisons of allozyme variation of narrow endemic and widespread species of Far East Euphorbia (Euphorbiaceae)/ Ki-Ryong Park
Phenotypic plasticity of Mosla chinensis and M. scabra (Labiatae) response to soil water status/ Bao-Hua GuanYing GeJie Chang
The occurrence of extrafloral nectaries in Hong Kong plants/ May-Ling So
Heat shock proteins of thermophilic and thermotolerant fungi from Taiwan/ Kuei-Yu ChenZuei-Ching Chen
Begonia coptidifolia (Begoniaceae), a new species from China/ Hua-Gu YeFa-Guo WangYu-Shi YeChing-I Peng
 
   
 
   

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