Identification and characterization of a rice cysteine endopeptidase that digests glutelin
Identification and characterization of a rice cysteine endopeptidase that digests glutelin
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DOI：
10.1111/j.1432-1033.1996.0310u.x
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发表时间：
1996-07-15
期刊：
影响因子：
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通讯作者：
Minamikawa, T
中科院分区：
文献类型：
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作者：
Kato, H;Minamikawa, T
在吸胀后第6天，暗栽水稻种子的贮藏器官提取液中几乎没有检测到内肽酶活性，吸胀后第9天，每粒种子表达的内肽酶活性显著增加，第18天达到最大值，然后下降。从第9天萌发的种子中，40-75%的饱和硫酸铵组分中存在两种主要的内肽酶：rep-1和rep-2，并可通过疏水柱层析进行分离。将Rep-1进一步纯化为36 kDa的单一多肽。REP-1在体外消化了水稻谷蛋白的酸性和碱性亚基，谷蛋白是水稻的主要种子储存蛋白。N-末端氨基酸序列测定和酶抑制剂实验表明，rep-1是一种半胱氨酸内肽酶。采用水稻种子文库筛选和5‘端快速扩增技术相结合的方法，测定了全长rep-1基因的核苷酸序列。
Little or no endopeptidase activity was detected in extracts from storage organs of dark-grown rice seeds until day 6 of post-imbibition, and the activity expressed per seed increased notably after day 9, reached a maximum on day 18, then decreased. Two major endopeptidases, REP-1 and REP-2, were present in the 40-75% saturated ammonium sulfate fraction from day-9 germinated seeds, and could be separated by hydrophobic column chromatography. REP-1 was further purified to a single polypeptide of 36 kDa. REP-1 digested in vitro both the acidic and basic subunits of rice glutelin, the major seed storage protein of rice. Determination of the N-terminal amino acid sequence and experiments with protease inhibitors indicated that REP-1 is a cysteine endopeptidase. The nucleotide sequence of a full-length REP-1 cDNA was determined by a combination of screening of cDNA libraries from rice seeds and the 5' rapid amplification of cDNA ends technique.