Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.
Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.
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DOI：
10.1083/jcb.96.3.802
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发表时间：
1983-03
期刊：
影响因子：
--
通讯作者：
Akazawa T
中科院分区：
文献类型：
--
作者：
Miyata S;Akazawa T
在小麦胚芽提取物（S-100级分）中孵育从水稻种子盾片制备的微粒体，以指导α-淀粉酶的合成，α-淀粉酶是一种分泌蛋白，在其生物合成过程中进行蛋白水解加工（N-末端信号序列的切割）以及糖基化。通过SDS凝胶电泳和随后的荧光法对合成的免疫沉淀产物进行表征和鉴定。由微粒体合成的α-淀粉酶的分子量被认为是相同的成熟分泌形式的酶的电泳迁移率的基础上。合成的酶分子的显著部分被证明被分离到微粒体囊泡中，并被保护免受内切-β-N-乙酰氨基葡萄糖苷酶的消化，这表明前体多肽链的蛋白水解加工和糖基化都发生在微粒体中。通过用Triton X-100破坏微粒体膜进一步检查多肽链的修饰。在蛋白质合成之前，微粒体的去污剂处理引起多肽链的蛋白水解加工和糖基化的抑制，导致除了α-淀粉酶的成熟形式之外，还合成未加工的新生（前体I）、加工的但非糖基化的新生（前体II）形式。此外，Triton X-100对多肽链修饰的抑制作用的时间序列分析结果使我们得出结论，在α-淀粉酶的生物合成期间，微粒体中发生蛋白水解加工和随后的糖基化。
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.