Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice
Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice
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DOI：
10.1007/s004250050560
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发表时间：
1999-04-01
期刊：
影响因子：
4.3
通讯作者：
Nakamura, Y
中科院分区：
文献类型：
--
作者：
Fujita, N;Kubo, A;Nakamura, Y
采用Q Sepharose HP阴离子交换层析、硫酸铵分离、TSKgel G4000SW（XL）和G3000SW（XL）凝胶过滤层析，在1天内通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对水稻（Oryza sativa L.）胚乳中的异淀粉酶（EC 3.2.1.68）进行了高效纯化。虽然该蛋白在SDS-PAGE上的分子量约为83 kDa，但在TSKgel G3000SW（XL）和G4000SW（XL）凝胶过滤层析上的表观大小分别约为340和490 kDa，这表明水稻同淀粉酶在发育中的胚乳中以四聚体到六聚体的形式存在。纯化后的水稻异淀粉酶能对糖原、植物糖原和支链淀粉进行脱枝，但不能对普鲁兰进行攻击。pH为6.5 ~ 7.0，温度为30℃时，对异淀粉酶的活性有较好的影响。该酶的活性在1 mM处被HgCl2和对氯喹苯甲酸盐完全抑制。这些结果表明，水稻同淀粉酶具有与报道的细菌同淀粉酶不同的特性。水稻胚乳同淀粉酶的互补dna克隆由玉米糖-1异淀粉酶cDNA保守核苷酸设计的引物聚合酶链反应产物作为探针（M.G. James et al., 1995, Plant Cell 7）。[J] .安徽农业大学学报（自然科学版），2003,(2):1- 7。最长无性系的核苷酸序列和推导出的氨基酸序列与玉米外科-1型异淀粉酶的相似性较高，与淀粉样假单胞菌异淀粉酶的相似性较低。Southern blot分析和基因定位分析表明，异淀粉酶基因在水稻基因组中以单拷贝形式存在，位于水稻第8染色体上。日本水稻属于粳稻组。系统发育分析表明，与假单胞菌和黄杆菌属的异淀粉酶相比，玉米和水稻的异淀粉酶与蓝绿藻和多种细菌的glgX基因产物的亲缘关系更密切。因此，有人提出glgX蛋白可归类为同淀粉酶型脱支酶。我们的树还表明，所有来自植物和细菌的淀粉和糖原去分支酶都可以分为两种不同的类型，一种是异淀粉酶型，另一种是普鲁兰酶型。
Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW(XL) and G3000SW(XL) gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW(XL) and G4000SW(XL) gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 degrees C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7. 417-429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271-9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.