Purification and Characterization of a Cytosolic Starch Phosphorylase from Etiolated Rice Seedlings

水稻白化苗澱粉磷解酶之純化與生化性質研究

許仁弘(Jen-Hung Hsu) ； 楊健志(Chien-Chih Yang) ； 蘇仲卿(Jong-Ching Su) ； 李平篤(Ping-Du Lee)

《Botanical Bulletin of Academia Sinica》 45卷3期 (2004/07) Pp. 187-196

https://doi.org/10.7016/BBAS.200407.0187

摘要

澱粉磷解酶 (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酶 (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 K(下標 m)值為 2.1mM ；V(下標 max)值為 5.85 Umg^(-1)。在磷解方向上，對磷酸根之K(下標 m)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, G(下標 6~10)) 親和性低。另外，H-RSP 對於糊精之 K(下標 m) 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(上標 +)、Hg(上標 2+)、Zn(上標 2+)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。

關鍵字

澱粉磷解酶 ； 水稻 ； 葡聚醣 ； 糊精 ； 親和性層析

並列摘要

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 k(subscript m) value was 2.1 mM, and the V(subscript max) value was 5.85 U mg ^-1. In the phosphorolytic direction for orthophosphate, the k(subscript m) value was 3.8 mM. Phol has a higher affinity for amylopectin, glycogen, soluble starch and dextrin than for maltooligosaccharide (6 to 10 glucose units). In addition, the k (subscript m) 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 (superscript +), Hg(superscript 2+), and Zn(superscript 2+) also reduce the enzyme activity. However, thiol reagents activate Phol activity, suggesting that sulfhydryl-group(s) may be required for enzyme stability.

並列關鍵字

Affinity chromatography ； Dextrin ； Glucan ； Rice ； Starch phosphorylase