SEPARATION AND CHARACTERIZATION OF 2 ENDOPEPTIDASES FROM COTYLEDONS OF GERMINATING VIGNA-MUNGO SEEDS
SEPARATION AND CHARACTERIZATION OF 2 ENDOPEPTIDASES FROM COTYLEDONS OF GERMINATING VIGNA-MUNGO SEEDS
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DOI：
10.1104/pp.80.3.628
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发表时间：
1986-03-01
期刊：
影响因子：
7.4
通讯作者：
MINAMIKAWA, T
中科院分区：
文献类型：
--
作者：
MITSUHASHI, W;KOSHIBA, T;MINAMIKAWA, T
两种主要的内肽酶存在于萌发的绿豆种子子叶，聚丙烯酰胺凝胶电泳后的酶谱检测。在干燥种子的子叶中检测不到，但在萌发过程中其酶谱强度增加。然而，在离体子叶的孵育期间，活性仅显示出轻微的增加，持续5天。用Sephacryl S-200柱层析可将两种内肽酶分离。其中之一被发现是丝氨酸内肽酶判断苯甲基磺酰氟和二异丙基氟磷酸盐抑制。另一种是巯基内肽酶，因为它依赖于2-巯基乙醇，并受到亮抑酶肽、凝乳酶抑制素和抗痛剂的抑制。十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析表明，两种内肽酶消化绿豆种子球蛋白亚基的速度不同。丝氨酸酶首先消化56千道尔顿亚基，但巯基酶消化54千道尔顿肽比消化56千道尔顿肽更有效。丝氨酸-和巯基-内肽酶的组合的球蛋白的消化模式是类似的，使用粗酶提取物。
Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2-mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts.