Purification and Characterization of Sucrose Phosphate Synthase from Sweet Photato Tuberous Roots

甘藷塊根蔗糖磷酯合成酶的純化與性質分析

陳韋良(Wei-Liang Chen) ； 黃東健(Dong-Jiann Huang) ； 劉邦熙(Pang-His Liu) ； 王恒隆(Heng-Long Wang) ； 蘇仲卿(Jong-Ching Su) ； 李平篤(Ping-Du Lee)

《Botanical Bulletin of Academia Sinica》 42卷2期 (2001/04) Pp. 123-129

https://doi.org/10.7016/BBAS.200104.0123

摘要

蔗糖磷酯合成酶(Sucrose phosphate synthase,簡稱SPS)是蔗糖生合成的一個重要酵素。以台農57號甘藷塊根為材料，酵素粗抽後，經DEAE-Sephacel離子交換層析，24%PEG沉澱，ω-aminohexyl Sepharose 4B和EPLCMono-Q管柱層析進行酵素純化，得到純化倍率約40倍，回收率約1%的SPS。經原態PAGE分析沒得酵素分子量約540kDa，另以SDS-PAGE分析推測其單元體分子量約為130~140kDa，得知SPS可能由同質四元體組成。經醣染色，證實其可能為醣蛋白，且其pI值約5.29。甘藷塊根SPS并不會受G6P及Pi的異位調控，與光合組織所得者不同。而SPS對基質F6P及UDPG之Km值分別為5.3mM及31.1mM。鎂、鈣及亞錳離子等兩價金屬離子對此酵素有促進作用，而汞離子則抑制其活性。ATP,ADP,AMP,UTP,UDP,UMP會降低SPS的活性約40~50%。CTP,GTP,GDP對SPS的活性并不會產生影響。甘藷塊根SPS對於硫氫化合物很敏感，在低濃度(0.1mM)時-SH化合物對酵素有安定作用，但過高(10mM以上)則反而有明顯抑制作用。SPS在葡萄糖，葡萄糖胺，麥芽糖，和乳糖中活性會增加，然而SPS則被δ－葡萄糖酸內酯完全抑制，可能是酵素會形成不穩定的葡萄糖－酵素化合物之故。硫氫化合物之抑制劑PCMBS會隨著濃度增加而降低SPS的活性。而另一抑制劑Cibacron blue F3G-A為UDPG的類似物，可以對SPS和UDPG結合區產生抑制。

關鍵字

甘藷 ； 蔗糖磷酯合成酶 ； 蔗糖代謝 ； 酵素純化

並列摘要

Sucrose phosphate synthase (SPS) is one of the key enzymes in the sucrose biosynthesis pathway. SPS was purified 40 fold from crude extract of sweet potato tuberous roots by the methods of batch elution from DEAE-Sephacel, PEG precipitation, ω-aminohexyl Sepharose 4B affinity and Mono Q anion exchange chomatographies. The native- and SDS-PAGE analyses revealed SPS to have a native molecular mass of about 540kDa, and it may therefore be homotetramer composed of subunit with a mass of 130-140kDa. The isoelectric point of the purified enzyme as determined by IEF was 5.29. SPS from the sweet potato tuberous root, which differs from the SPS of photosynthetic tissues, was not allosterically regulated by G6P and Pi. The Km for F6P and UDPG was 5.3 and 31.3mM, respectively. The enzyme was activated by Mn (superscript 2+), Mg (superscript 2+), and Ca (superscript 2+), while being inhibited by Hg (superscript 2+). The nucleotides AMP, ADP, ATP, UMP, UDP, UTP, and TDP inhibited the enzyme about 30~50%. The enzyme was sensitive to sulfydryl reagents, but activity could be restored with DTT or β-ME. The enzyme was activated by glucose, glucosamine, maltose, and lactose, but was inhibited by δ-gluconolactone. SPS could also be inhibited by PCMBS and Cibacron blue F3G-A.

並列關鍵字

Enzyme purification ； Ipomoea batatas ； Sucrose metabolism ； Sucrose phosphate synthase ； Sweet potato