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绿豆胰蛋白酶抑制剂的研究——Ⅷ.两个活性碎片的拆分

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Acta Biochimica et Biophysica Sinica 1982年第2期 35-42页

作者：谭复隆戚正武中国科学院上海生物化学研究所

1.绿豆胰蛋白酶抑制剂有二个活性中心,可以同时抑制两分子胰蛋白酶。用苯乙二醛及顺丁烯二酸酐分别进行化学修饰,都可使其活力降低至原有的50%左右,表明此两活性中心分别应为精氨酸残基及赖氨酸残基。2.绿豆抑制剂经胃蛋白酶酶解后,活... 详细信息

1.绿豆胰蛋白酶抑制剂有二个活性中心,可以同时抑制两分子胰蛋白酶。用苯乙二醛及顺丁烯二酸酐分别进行化学修饰,都可使其活力降低至原有的50%左右,表明此两活性中心分别应为精氨酸残基及赖氨酸残基。2.绿豆抑制剂经胃蛋白酶酶解后,活力不丧失,在凝胶过滤中出现分子量为原抑制剂一半的新活力峰,经证实为两个不同活性中心的活力碎片。3.利用两活性中心残基赖氨酸和精氨酸侧链基团解离pK 值的差异,在pH11.4的条件下通过固相胰蛋白酶亲和层析可将两活力碎片彼此分离。分离所得两碎片活力大致相等。4.以赖氨酸为活性中心的碎片能被顺丁烯二酸酐全部抑制失活,在pH3.5下保温却和原抑制剂一样能恢复其原有活力的90%以上。此活力碎片由两条肽链所组成,共含35个氨基酸残基,N-末端为丝氨酸和苯丙氨酸,两C-末端分别为亮氨酸及甲硫氨酸。5.以精氨酸为活性中心的碎片的活力能被苯乙二醛全部抑制,经鉴定为一条肽链,其含约27个氨基酸残基,N-末端为门冬酰胺,C-末端为门冬氨酸。

关键词：活性中心绿豆胰蛋白酶抑制剂碎片活性区域拆分

STUDIES ON NATURAL AND MODIFIED PEPTIDE Trichosanthes TRYPSIN INHIBITORS

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Science China Chemistry 1990年第5期33卷 599-605页

作者：钱岳伟谭复隆戚正武 Shanghai Institute of Biochemistry Academia Sinica Shanghai 200031 PRC

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C_(18) column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3--4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.

关键词： Trichosanthes trypsin inhibitor enzyme modification amino acid sequence.

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ACTIVE DOMAINS IN MUNG BEAN TRYPSIN INHIBITOR

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Science in China,Ser.B 1982年第2期25卷 160-167页

作者：谭复隆戚正武 Shanghai Institute of Biochemistry Academia Sinica

In our experimentes, it has been demonstrated that the N-terminal fragment of the mungbean inhibitor could be isolated by affinity chromatography with immobilized trypsin aftertreating the inhibitor with CNB_r and pepsin. The properties of this active fragment have beenstudied. The C-terminal fragment of the hihibitor, however, will lose its activity during CNB_rcleavage because of the scission of the methionyl peptide bond near the active center. It isdeemed desirable to find out a more suitablo method for the cleavage of these two domains,both retaining inhibitory activity. Ths aim has new been achieved by using peptic digestion only, and the active centers ofthese two domains are identified as Lys and Arg by chemical modification. In alkaline pH,these two active fragments could be easily separated by affinity chromatography with immobi-lized trypsin. This amkes it possible to study and compare these two active fragmentsseparately. It is worth pointing out that such a C-terminal fragment with trypsin inhibitory activityhas not yet been reported among the Bowman-Birk inhibitors. The activities of the mung bean trypsin inhibitor decrease to about 50% during thecourse of modification with maleic anhydride and phenylglyoxal respectively. The two activecenters seem to be Lys nd Arg. The mung bean inhibitor could endure peptic digestionwithout any loss of activyty. On Sephadex gel filtration, two smaller active fragments with asize approximately one half of the original molecule are found. At pH 11.4 these two activefragments could be separated from each other by affinity chromatography with immobilizedtrypsin. The active fragment with Lys as the active center could be completely inhibitedwith maleic anhydride and the activity is completely restored after incubation at pH *** fragmeut consists of two paptide chains with about 35 amino acil residues. The N-termini are found to be Ser and Phe, and the C-termini to be Leu and Met. The activityof the fragment with Arg

关键词： ACTIVE DOMAINS IN MUNG BEAN TRYPSIN INHIBITOR

绿豆胰蛋白酶抑制剂的研究——Lys活性碎片的还原及重氧化

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中国科学:化学 1984年第8期 717-723页

作者：谭复隆施庆洛王丽秀戚正武中国科学院上海生物化学研究所中国科学院上海脑研究所

本文证明绿豆胰蛋白酶抑制剂Lys活性碎片由 A,A两条肽链通过两对二硫键联结而成,今通过DTT还原及凝胶过滤可将A（26个氨基酸残基）,A（9个氨基酸残基）两肽链拆分.还原型的A肽链重氧化后活力的回收率是原有活性碎片总活力的25%.经固相... 详细信息

本文证明绿豆胰蛋白酶抑制剂Lys活性碎片由 A,A两条肽链通过两对二硫键联结而成,今通过DTT还原及凝胶过滤可将A（26个氨基酸残基）,A（9个氨基酸残基）两肽链拆分.还原型的A肽链重氧化后活力的回收率是原有活性碎片总活力的25%.经固相胰蛋白酶亲和层析纯化的A活性碎片以单体和二聚体两种分子形式存在,两者可在Sephadex G-25柱上分开,以单体为主,二聚体活性较低.此结果进一步证实,绿豆胰蛋白酶抑制剂的分子进化过程是由一原始单头抑制剂通过基因重复及基因突变演化而来。此外比较了Lys活性碎片和重氧化肽链A的CD图谱。

关键词： Lys 绿豆胰蛋白酶抑制剂活性区域碎片

COMPLETE AMINO ACID SEQUENCE OF MUNG BEAN TRYPSIN INHIBITOR

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Science China Chemistry 1982年第3期25卷 268-277页

作者：张耀时罗珊珊谭复隆戚正武徐烈贤张爱乐 Shanghai Institute of Biochemistry. Academia Sinica Nanjing University Vopartment of Biochemistry

The mung bean trypsin inhibitor has been found to be microheterogeneous at N-terminalregion due to the presence of several isomers. After treatment with aminopeptidase M it becomeshomogeneous and is suitable for sequence determination. Based on the determination of the structures of two active fragments the complete aminoacid sequence of mung bean trypsin inhibitor has been elucidated. It consists of 72 amino acidresidues with 7 pairs of disulfide bonds. The results show that this inhibitor belongs to theBowman-Birk inhibitor family.

关键词： Pro CysC COMPLETE AMINO ACID SEQUENCE OF MUNG BEAN TRYPSIN INHIBITOR

STUDIES ON THE MUNG BEAN TRYPSIN INHIBITOR——REDUCTION AND REOXIDATION OF THE DISULFIDE BONDS OF THE LYS ACTIVE FRAGMENT

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Science in China,Ser.B 1984年第9期27卷 918-925页

作者：谭复隆施庆洛王丽秀戚正武 Shanghai Institute of Biochemistry Academia Sinica

Two peptide chains A1 and A2 of the Lys active fragment, linked via a couple ofinter-disulfide bonds, could be separated from each other after reduction with dithiothreitoland gel filtration on Sephadex G-25. Reoxidation of the reduced peptide chain A1 resultedin recovering the inhibitory activity with 25% yield, based on the original activity of theLys fragment. The A1 active fragment was further purified by affinity chromatographywith immobilized trypsin. Sephadex G-25 gel filtration produced two forms of the A1 activefragment, the major fraction being a monomer and the minor one being a dimeer with loweractivity. The results obtained offered evidence of the evolution of mung bean inhibitorfrom an ancestral single-headed inhibitor by fused gene duplication with A2 as a connectingpeptide. The CD spectra of the Lys fragment and the reoxidized peptide chain A1 werealso compared.

关键词： REDUCTION AND REOXIDATION OF THE DISULFIDE BONDS OF THE LYS ACTIVE FRAGMENT STUDIES ON THE MUNG BEAN TRYPSIN INHIBITOR

绿豆胰蛋白酶抑制剂Lys活力碎片-牛胰蛋白酶复合物的晶体学研究

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中国科学:化学 1986年第3期 273-275页

作者：张荣光林光大严友为唐伟中谭复隆戚正武曹天钦中国科学院上海生物化学研究所

本文证明绿豆胰蛋白酶抑制剂的Lys活力碎片能当量结合胰蛋白酶形成复合物。用微量静置法得到该复合物的单晶。X射线晶体学研究表明晶体衍射分辨率可达1.8（?）,空间群为P222,晶胞参数a=62.9（1）(?),6=63.4（1）(?),c=69.7（2）(?)。每... 详细信息

本文证明绿豆胰蛋白酶抑制剂的Lys活力碎片能当量结合胰蛋白酶形成复合物。用微量静置法得到该复合物的单晶。X射线晶体学研究表明晶体衍射分辨率可达1.8（?）,空间群为P222,晶胞参数a=62.9（1）(?),6=63.4（1）(?),c=69.7（2）(?)。每个结晶学不对称单位含有一个复合物分子。

关键词：复合物单晶晶形胰蛋白酶浮选药剂抑制剂碎片晶体学研究 Lys

绿豆胰蛋白酶抑制剂及其酶复合物的晶体生长和X-射线晶体学参数测定

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科学通报 1985年第4期30卷 319-320页

作者：林光大张荣光梁栋材戚正武唐伟中王丽秀谭复隆曹天钦中国科学院生物物理研究所中国科学院上海生物化学研究所北京中国科学院上海生物化学研究所

绿豆胰蛋白酶抑制剂属于Bowman Birk类抑制剂,MW约8000,有7对二硫键,其一级结构已经测定。它有两个活性区域,活性中心分别为Lys-Set及Arg-Ser,可以以1:2或1:1的克分子比和胰蛋白酶络合。经胃蛋白酶降解后的两个活性片段也已分离提纯。... 详细信息

绿豆胰蛋白酶抑制剂属于Bowman Birk类抑制剂,MW约8000,有7对二硫键,其一级结构已经测定。它有两个活性区域,活性中心分别为Lys-Set及Arg-Ser,可以以1:2或1:1的克分子比和胰蛋白酶络合。经胃蛋白酶降解后的两个活性片段也已分离提纯。这一切为开展X-射线晶体结构测定提供了有利条件。在以往对绿豆抑制剂及其酶复合物能形成晶体

关键词：复合物射线晶体学晶体生长绿豆胰蛋白酶抑制剂衍射分辨率不对称单位克分子比

THE CRYSTALLOGRAPHIC STUDY OF THE COM PLEX OF THE LYS ACTIVE FRAGMENT OF THE MUNG BEAN TRYPSIN INHIBITOR WITH BOVINE TRYPSIN

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Science in China,Ser.B 1985年第11期28卷 1163-1166页

作者：张荣光林光大严友为唐伟中谭复隆戚正武曹天钦 Shanghai Institute of Biochemistry Academia Sinica

The Lys fragment of mung bean trypsin inhibitor can combine with bovine trypsin to form a complex at an equal molar ratio. The single cxystals of the complex were obtained by using the micro-still-setting method and the X-ray diffraction extended to 1.8 resolution. Its space group is P212121 wlth cell dimensions α=62.9（1）, b=63.4（1） and c=69.7（2）. There is one complex molecule in a crystallographic asymmetric unit.

关键词： THE CRYSTALLOGRAPHIC STUDY OF THE COM PLEX OF THE LYS ACTIVE FRAGMENT OF THE MUNG BEAN TRYPSIN INHIBITOR WITH BOVINE TRYPSIN LYS