摘要
从杜仲（ＥｕｃｏｍｍｉａｕｌｍｏｉｄｅｓＯｌｉｖ）皮中分离纯化到一种新的抗真菌蛋白（ＥｕｃｏｍｍｉａＡｎｔｉｆｕｎｇａｌＰｒｏｔｅｉｎ），简称ＥＡＦＰ。将切碎的杜仲树皮用ＮａＣｌ溶液在组织捣碎器中高速匀浆提取，经硫酸铁沉淀、ＣＭ一ｃｅｌｌｕｌｏｓｅ离子交换层析和Ｂｉｏ一ｇｅｌ一ｐ一１０凝胶层析纯化。如此纯化而得的ＥＡＦＰ在ＳＤＳ－ＰＡＧＥ胶片上显示一条分子量为５．０ｋＤ的单一带。反相ＨＰＬＣ显示４样品是纯的。等电聚焦电泳测得其ｐＩ值大于１１．０。经还原和氨酰羧甲基化处理，用ＨＰＬＣ层析证明ＥＡＦＰ为单链。用酚一硫酸法未测出其含糖。ＥＡＦＰ是简单单链蛋白。氨基酸组成分析结果表明它宫含Ａｒｇ、Ｃｙｓ和Ｇｌｙ，不含Ｍｅｔ，Ｌｙｓ，Ｔｒｐ，Ｈｉｓ和Ｐｈｅ。未经热变性或ＳＤＳ处理的ＥＡＦＰ与考马氏亮蓝试剂不发生显色反应。手工Ｅｄｍａｎ降解法测得Ｎ一端３个氨基酸残基的顺序为Ｇｌｙ一Ａｓｐ一Ａｌａ。用羧肽酶Ａ和Ｂ酶解ＥＡＦＰ测得其Ｃ一末端为Ｖａｌ．０．３ｍｇ／ｍＬ蛋白明显抑制木霉、小麦赤霉菌、烟草黑茎病菌和棉花枯萎病菌菌丝的生长。蛋白在沸水中保温３０ｍｉｎ活性不变。
A new antifugal protein named EAFP was isolated with 0.5mol /L NaCl from the bark pieces of Eucommia ulmoides Oliv and purified by ammonium sulfate fractionation,carboxymethylcellulose ion一exchange chromatography and Bio-gel一p-10 gel filtration. Purified EAFP thus abtained gave a single band having a molecular weight of 5 kD on SDS / PAGE. An elution pattern of the sample in reverse phase HPLC indicated that the sample was Dure. EAFP is a single polypeptide and pI> 11.0 determined by reduction and S一carboxamidomethylation and eletrofocusing on Ampholine/polyacrylamide一gel plates. There is not carbohydrate in EAFP detected by phenol一sulfate reaction. Analysis of the composition of amino acids showed that EAFP is rich in Arg,Cys and Gly, but lacks Met,Lys,Trp,His and Phe. EAFP can not give col-our reaction with Coomassie blue reagent except that the protein is denatured by heat or SDS treatment.Manual Edman degradation showed that sequence of EAFP from N一tenninus was Gly一Asp一Ala一and the digestion with carboxypeptidase A and B showed that C一terminal residue of EAFP was Val.EAFP at 0.3mg/mL inhibited obviously the growth of Trichoderma viride Pers:Fr.mycelia and some crop fungal pathogens.The antifungal activity of EAFP was unchanged by incubation at boiling temperature.
出处
《云南植物研究》
CSCD
1994年第4期385-391,共7页
Acta Botanica Yunnanica
基金
国家63计划资助
关键词
杜仲
抗真菌蛋白
分离
Eucommia ulmoldes, Antifungal protein
