綠豆澱粉合成酵素的研究--酵素鑑定與生化性質的探討（II）

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Title:	綠豆澱粉合成酵素的研究--酵素鑑定與生化性質的探討（II） Study of Mungbean (Vigna radiata) Starch Synthases---Biochemical Properties Enzyme Characterization (II)
Authors:	柯源悌
Contributors:	中國醫藥大學營養學系
Keywords:	綠豆;澱粉合成酵素;酵素化學;Mungbean;Starch synthase;Enzyme chemistry
Date:	2002-07
Issue Date:	2010-07-16 11:42:45 (UTC+8)
Abstract:	綠豆(mungbean; Vigna radiata cv. KPS1)萃取液(B7 crude)經 Native-PAGE，再以磷解刺激的澱粉分支酵素(starch branching enzyme; SBE)之原位活性染色反應，會同時出現一電泳泳動慢的藍紫色(SBEI)與兩個電泳泳動快的紅紫色(SBEIIa，IIb)蛋白質族群。且SBEI於反應2小時即會出現，但SBEIIa要16小時後才會出現。SBEI和SBEIIa族群經SDS-PAGE及銀染得知分別主要包含 105、62和55 kDa及96、64和55 kDa的蛋白質。B7 crude經一連串的部份純化，發現通過Sephacryl S-200管柱時，會分離得到PI和 PII兩個尖峰，且由活性染色實驗得知所分離出的PI是屬於活染為藍紫的SBEI族群，而分離出的PII是屬於紅紫的SBEIIs族群。最後分別經過Q2 管柱，得到純化倍數為30.4倍的PIII和101.9倍的PIV。另外，由電泳圖發現SBEI族群包含的105、62和55 kDa蛋白質之含量與活性有量相關性，並且也發現SBEIIa族群包含的96、64和55 kDa蛋白質之含量亦有量相關性。105 kDa蛋白質經過trypsin水解與蛋白質質譜鑑定得知與地瓜、玉米、稻米…等有極高的相似度，推測為綠豆之澱粉磷解酵素(starch phosphorylase; SP)。以62 kDa蛋白質製備的抗體，來進行免疫中和反應，發現抗體能中和B7 crude中的SBE 活性達97﹪；且於西方點墨法發現此抗體主要可辨識到62、55 和 31 kDa的蛋白質。而 55 kDa的抗體亦可中和B7 crude且辨識到62、55 和 31 kDa的蛋白質。因此，推斷62和55 kDa蛋白質是屬於SBEI族群，其會與可能為SP的105 kDa蛋白質在原始態下相互結合。當三種不同發育時期綠豆種子之粗萃取液，以磷解刺激SBE之活性染色分析，發現顆粒大的種子其SBE比活性會減小，且於活染顯影知道SBEI族群會減少而SBEIIa族群反而會增加，但是DS-PAGE 顯示SBEI族群之主要蛋白質的分子量相同，包含62 及 55 kDa，含量也相類似。由以上結果可知，至少有三種不同的SBE異構在綠子發育過程中，對澱粉的生合成扮演不同的重要角色。 Partially purified mungbean ( Vigna radiata var. KPS1) soluble fractions with starch branching enzyme (BE) activity detected by radioactive method were subjected to Native-PAGE and in situ activity staining for enzyme analysis. When native gel was incubated with G-1-P and sodium citrate (pH 7.0) to react at 30 ？HJ for 2 hrs followed by iodine staining, it visualized a blue-purple protein population with slow mobility. SDS-PAGE and silver staining showed that it was consisted of 105, 62 and 55 kDa proteins. The polyclonal antibody against the 62 kDa protein was able to immuno-neutralize 97% SBE activity and cross-react majorly with 62, 55 and 31 kDa proteins of mungbean crude extract by Western blotting. The polyclonal antibody against the 55 kDa protein also cross-reacted with 62, 55 and 31 kDa proteins. Tryptic peptide mapping and database searching for the 105 kDa protein demonstrated it to be low-affinity starch phosphorylase (L-SP) due to its high internal sequence homology with L-SP of sweet potato, corn, rice and potato. It suggested that mungbean L-SP is associated with 62 kDa and 55 kDa protein at native state. When native gel was incubated in the presence of phosphorylase a and AMP in addition to G-1-P and sodium citrate (pH 7.0) to react at 30 ？HJ for 21 hrs, two red-purple protein populations with high mobility were visualized by iodine staining, which was similar to the finding of rice BEIIa and BEIIb. Developing mungbeans of different seed size were analyzed by the phosphorylase-stimulated activity staining method and found that the density of SP-containing slow mobile population decreased and the high mobile population increased as the seed size increase. These results demonstrated that various mungbean BE isoforms may play important roles in starch accumulation during seed development.
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