Construction of chimeric genes for elusidating unique properties of amylase-III

从水稻培养细胞中分离并鉴定了两种α-淀粉酶同工酶，分别命名为AMY-I和AMY-III。其中AMY-III的低温最适温度为25ﾟC，与包括AMY-I在内的其他植物α-淀粉酶不同，该酶一般有50ﾟC左右。为了了解AMY-III的这些独特性质的原因，我们分离并鉴定了AMY-I和AMY-III的cDNA克隆，并研究了这些克隆及其与AMY-I和AMY-III的不同组合在酿酒酵母中的表达。根据周和法斯曼的方法对AMY-III的二级结构分析表明，位于相关区域B和C之间的区域-C为b-折叠结构，另一方面，AMY-I和其他研究较多的淀粉酶，即稻米曲霉淀粉酶(TAA)、猪胰腺淀粉酶(PPA)都显示了该区域的a-螺旋结构。事实上，从携带pGMlu III I、pGSca III I或pGSal I III的酵母中获得的嵌合体蛋白都具有AMY-I的c区域的特征，这些都是分泌性蛋白。相反，从携带pGNae III I、pGMlu I III或pGSca I III的酵母中获得的其他类型的嵌合体蛋白在培养基中不分泌，免疫印迹分析表明，从携带载体pG-3的酵母细胞中获得的对AMY-III的迁移率与从携带AMY-III的PG-3酵母细胞制备的相同。这表明AMY-III和AMY-I-AMY-III嵌合体蛋白在酵母细胞内积累。这些结果表明，AMY-III蛋白的c区决定了AMY-III的特性。

From cultured-cells of rice (Oryzae sativa cv Sasanishiki), we previously isolated and characterized two a-amylase isozymes (designated as AMY-I and AMY-III). One of them, AMY-III,showed a low temperature optimum of 25ﾟC unlike that for other plant a-amylases including AMY-I,thet generally had around 50ﾟC.To know what is responsible for these unique properties of the AMY-III,we isolated and characterized cDNA clones for AMY-I and-III,and studied the expression of those and the chimeric genes with various combinations of AMY-I cDNA and AMY-III cDNA in Saccharomyces cerevisiae.Secondary structure analysis of AMY-III according to the method of Chou and Fasman revealed that the region-c lying between the concerved regions B and C showed b-sheet structure, on the other hand, AMY-I and other well studied amylases, namely A.oryzae amylase (TAA), porcine pancreatic amylase (PPA) have shown a-helix structutre for this region In fact, chimera protiens obtained from the yeasts carrying a plasmid of pGMlu III I or pGSca III I or pGSal I III,all of which contain region-c of AMY-I showed characteristic properties of AMY-I,and these were all secreted proteins. On the contrary, other type of chimera proteins obtained from the yeasts carrying a plasmid of pGNae III I or pGMlu I III or pGSca I III,all of which contain region-c of AMY-III,were not secreted in the medium and immunoblotting analysis showed the same mobility to AMY-III that prepared from yeast cells carrying a vector, pG-3 with AMY-III.This indicates AMY-III and AMY-I-AMY-III chimera proteins carrying region-c of AMY-III accumulate intracellularly in yeast. These results suggest that the region-c of AMY-III protein determines the characteristic properties of AMY-III.