萌发花生种子子叶肽链内切酶的纯化和性质

PURIFICATION AND CHARACTERIZATION OF CYSTEINE ENDOPEPTIDASE IN COTYLEDONS OF GERMINATED PEANUT (ARACHIS HYPOGAEA L.) SEEDS

摘要:

萌发花生种子子叶的肽链内切酶经硫酸铵沉淀，ＳｅｐｈａｄｅｘＧ－１００凝胶层析，ＤＥＡＥ－纤维素２３阴离子交换层析和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ５０层析，得到纯化的酶，该酶有两条同工酶，分子量分别为５８和５５ＫＤ，Ｋｍ为９．９μｍｏｌ／Ｌ，是半胱氨型肽链内切酶（ＥＣ３．４．２２），对未萌发花生种子的贮藏蛋白没有明显降解作用．

Abstract:

A cysteine endopeptidase (EC 3.4.22) was purified from cotyledons of germinated peanut seeds by buffer extraCtion, ammonium sulfate precipitation and succesive chromatagrafies on Sephadex G-100, DEAE-cellulose and DEAE-Sephedax A-50 columes. The overall purification and final recovery were 28.1 fold and 15.9%. This endopeptidase exists in two isoenzymic forms and SDS-PAGE revealed two polypeptides of 58 and 55KD. The optimumtemperature was 50℃ at 8.1 pH for degrating BAPNA (Benzoylarginine-p-nitroanilide) in Tris-HCl buffer. Inhibitor studies demonstrated that the endopeptidase belongs to the cysteine class of endopeptidase. This enzyme hydrolysed only 5-10% of arachin or coarachin Ⅰor 2s protein of ungerminated peanut seeds in vitro. The characterization of the endopeptidase was almost consistent with the peptide hydrolase in the study of Cameron and Mazelis who purificated from cotyledons of ungeednated peanut seeds.The results of present paper and our other studies suggested that the cysteine endopeptidase of peanut cotyledons which plays an important role during seed germination was synthesized during seed development and can only degrade the modified storge proteins.