Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha-amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the alpha-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-beta-N-acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor II) forms, in addition to the mature form of alpha-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of alpha-amylase.
Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.
S Miyata, T Akazawa; Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.. J Cell Biol 1 March 1983; 96 (3): 802–806. doi: https://doi.org/10.1083/jcb.96.3.802
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