Abstract
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.
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