Miyata S, Akazawa T
Proc Natl Acad Sci U S A. 1982 Dec;79(24):7792-5. doi: 10.1073/pnas.79.24.7792.
Recently, we showed that completion of the polypeptide chains on the polysomes isolated from germinating rice seed scutellum in a cell-free translation system can direct the synthesis of (i) unprocessed polypeptide containing the signal sequence (precursor 1), (ii) signal sequence-cleaved but nonglycosylated polypeptide (precursor 2), and (iii) the fully processed and glycosylated form of alpha-amylase molecules. The two precursors as well as the mature form of alpha-amylase can thus be produced in almost the same condition. Here, the binding affinity of the enzyme molecule to the substrate analogue beta-cyclodextrin was used as a probe to compare the conformations of the three distinctly different polypeptide chains produced on the polysomes. It was found that the mature secretory form and the nonglycosylated precursor form (precursor 2) specifically bind to beta-cyclodextrin immobilized on an epoxy-activated Sepharose 6B column but the form that has an attached signal sequence (precursor 1) does not. The results provide evidence that the NH2-terminal signal sequence prevents acquisition of beta-cyclodextrin-binding activity, indicating that, in rice seed alpha-amylase, the signal sequence impairs conversion of the unprocessed polypeptide to the enzymically active configuration.
最近，我们发现，在无细胞翻译系统中，从萌发的水稻种子盾片中分离出的多核糖体上完成的多肽链能够指导合成：（i）含有信号序列的未加工多肽（前体1），（ii）信号序列被切割但未糖基化的多肽（前体2），以及（iii）α-淀粉酶分子的完全加工和糖基化形式。因此，在几乎相同的条件下可以产生这两种前体以及α-淀粉酶的成熟形式。在此，利用酶分子与底物类似物β-环糊精的结合亲和力作为探针，比较在多核糖体上产生的三种明显不同的多肽链的构象。结果发现，成熟的分泌形式和未糖基化的前体形式（前体2）能特异性结合固定在环氧活化的琼脂糖6B柱上的β-环糊精，而带有附着信号序列的形式（前体1）则不能。这些结果提供了证据，表明NH2末端信号序列阻止了β-环糊精结合活性的获得，这表明，在水稻种子α-淀粉酶中，信号序列会损害未加工多肽向酶活性构象的转化。
