The structure of the complex of mung bean trypsin inhibitor lysine active fragment with bovine trypsin has been determined at a resolution of 1.8 A by A-ray crystallographic analysis and the complex model refined by r...The structure of the complex of mung bean trypsin inhibitor lysine active fragment with bovine trypsin has been determined at a resolution of 1.8 A by A-ray crystallographic analysis and the complex model refined by restrained least-squares minimization with the data between 10 and 1.8 resolution.The current conventional R factor is 17.3%,and the model con- tains 1648 protein atoms,219 inhibitor atoms and 126 water molecules.The most prominent feature of the inhibitor fragment is that it does not contain any alpha-helices.Most of the chain fold in an irregular fashion.The seven residues of the binding segment of the inhibitor lysine active frag- ment are in specific contact with bovine trypsin.The binding interaction and geometry around the reactive site are similar to that observed in other studies of trypsin-inhibitor complexes.展开更多
The Lys active fragment of the mung bean trypsin inhibitor is composed of two peptidechains,one with 26 amino acid residues and the other with 9 residues linked by two interdisulfide bonds.The two peptide chains could...The Lys active fragment of the mung bean trypsin inhibitor is composed of two peptidechains,one with 26 amino acid residues and the other with 9 residues linked by two interdisulfide bonds.The two peptide chains could be separated successfully from each other by reduction.with DTT followedby gel filtration.The reduced long peptide chain containing 6 Cys residues was subjected to air oxidation,and about 25% of the original antitrypsin activity of the Lys fragment was recovered.Following the previ-ously reported sequence,the solid-phase synthesis of this long peptide chain and its disulfide bond refold-ing are presented.Unexpectedly,the synthetic peptide showed much lower antitrypsin activity than thenatural one after reduction and air reoxidation.In order to explain this uncompatible result,we redeter-mined the sequence of the native long peptide chain of the Lys active fragment and obtained the result thatthe P′<sub>2</sub> position is Ile instead of Lys as previously reported.To ascertain the correct sequence,we synthe-sized another 22-peptide following the newly determined 26-peptide sequence,and skimming two residuesrespectively from N-terminus and C-terminus.After reduction and reoxidation,the synthetic 22-peptidehad the same antitrypsin activity as that of the native 26-peptide.Meanwhile,an analogue of this 22-pep-tide in which the residue Lys at the reactive site was replaced by Ala was also synthesized.This syntheticanalogue did not show any activity either to trypsin or to elastase.展开更多
By 30%～60%s(NH\-4)\-2SO\-4 fractional precipitation,anion\|exchange chromatographs on DEAE\|Sepharose CL\|6B,gel filtration on Sephacryl S\|200 and anion\|exchange chromatographs on Waters AP\|1 column(Protein Tm \|P...By 30%～60%s(NH\-4)\-2SO\-4 fractional precipitation,anion\|exchange chromatographs on DEAE\|Sepharose CL\|6B,gel filtration on Sephacryl S\|200 and anion\|exchange chromatographs on Waters AP\|1 column(Protein Tm \|Pak DEAE 15HR),a proteinase which can inactivated STI was purified from mung bean( Phaseolus aureus )burgeon.It was stable at temperatures lower than 50℃ and pH7.5～8.5,and the K m and V max of the proteinase for STI was 769.2 α\|N\|benzoyl\|L\|arginine ethyl ester(BAEE)/mL and 115.3BAEE/min/mL respectively.The molecular weight of the proteinase was estimated to be 29.8kD by SDS\|PAGE.The proteinase immobilized by polyacrylamide was stable at temperatures lower than 60℃ and pH7.0～9.0,and the apparent K * m and V max * of the immobilized proteinase for STI was 1303.8(BAEE)/mL and 94.34(BAEE)/min/mL respectively.The half\|life of the immobilized proteinase was about 12 days at展开更多
以4个抗豆象绿豆品系B18、B20、B24和A22为试材,以感虫绿豆品种晋绿1号为对照,研究了不同绿豆中胰蛋白酶抑制剂活性及其在高温、酸碱及超声波下绿豆的胰蛋白酶抑制剂稳定性。结果表明,4个抗豆象绿豆品种胰蛋白酶抑制剂活性均显著高于对...以4个抗豆象绿豆品系B18、B20、B24和A22为试材,以感虫绿豆品种晋绿1号为对照,研究了不同绿豆中胰蛋白酶抑制剂活性及其在高温、酸碱及超声波下绿豆的胰蛋白酶抑制剂稳定性。结果表明,4个抗豆象绿豆品种胰蛋白酶抑制剂活性均显著高于对照感虫品种,且均与对照在0.01水平差异极显著,其中B18活性最高,高达70.2TI U g–1,B20和A22活性次之,B24活性最差,但仍高达55.2 TI U g–1。4个抗豆象绿豆品种在不同温度、不同p H和不同振幅超声波下,残余活性均比对照高,且残余活性均随温度升高、温浴时间延长而降低,p H在2~12之间,随p H值的升高,残余活性均呈现先升高后降低的趋势,且p H值为6~8之间残余活性最高,残余活性也随超声波辐射强度升高、时间延长而降低,且4个抗虫品种中B18的耐高温性、耐酸碱性和耐辐射性最强,B20次之,B24的耐高温性、耐酸碱性最差,A22耐辐射性最差,说明在不同温度、p H和超声波处理后,B18、B20是抗豆象绿豆胰蛋白酶抑制剂残余活性保存最高的2个品种,应用价值较大。展开更多
文摘The structure of the complex of mung bean trypsin inhibitor lysine active fragment with bovine trypsin has been determined at a resolution of 1.8 A by A-ray crystallographic analysis and the complex model refined by restrained least-squares minimization with the data between 10 and 1.8 resolution.The current conventional R factor is 17.3%,and the model con- tains 1648 protein atoms,219 inhibitor atoms and 126 water molecules.The most prominent feature of the inhibitor fragment is that it does not contain any alpha-helices.Most of the chain fold in an irregular fashion.The seven residues of the binding segment of the inhibitor lysine active frag- ment are in specific contact with bovine trypsin.The binding interaction and geometry around the reactive site are similar to that observed in other studies of trypsin-inhibitor complexes.
文摘The Lys active fragment of the mung bean trypsin inhibitor is composed of two peptidechains,one with 26 amino acid residues and the other with 9 residues linked by two interdisulfide bonds.The two peptide chains could be separated successfully from each other by reduction.with DTT followedby gel filtration.The reduced long peptide chain containing 6 Cys residues was subjected to air oxidation,and about 25% of the original antitrypsin activity of the Lys fragment was recovered.Following the previ-ously reported sequence,the solid-phase synthesis of this long peptide chain and its disulfide bond refold-ing are presented.Unexpectedly,the synthetic peptide showed much lower antitrypsin activity than thenatural one after reduction and air reoxidation.In order to explain this uncompatible result,we redeter-mined the sequence of the native long peptide chain of the Lys active fragment and obtained the result thatthe P′<sub>2</sub> position is Ile instead of Lys as previously reported.To ascertain the correct sequence,we synthe-sized another 22-peptide following the newly determined 26-peptide sequence,and skimming two residuesrespectively from N-terminus and C-terminus.After reduction and reoxidation,the synthetic 22-peptidehad the same antitrypsin activity as that of the native 26-peptide.Meanwhile,an analogue of this 22-pep-tide in which the residue Lys at the reactive site was replaced by Ala was also synthesized.This syntheticanalogue did not show any activity either to trypsin or to elastase.
文摘By 30%～60%s(NH\-4)\-2SO\-4 fractional precipitation,anion\|exchange chromatographs on DEAE\|Sepharose CL\|6B,gel filtration on Sephacryl S\|200 and anion\|exchange chromatographs on Waters AP\|1 column(Protein Tm \|Pak DEAE 15HR),a proteinase which can inactivated STI was purified from mung bean( Phaseolus aureus )burgeon.It was stable at temperatures lower than 50℃ and pH7.5～8.5,and the K m and V max of the proteinase for STI was 769.2 α\|N\|benzoyl\|L\|arginine ethyl ester(BAEE)/mL and 115.3BAEE/min/mL respectively.The molecular weight of the proteinase was estimated to be 29.8kD by SDS\|PAGE.The proteinase immobilized by polyacrylamide was stable at temperatures lower than 60℃ and pH7.0～9.0,and the apparent K * m and V max * of the immobilized proteinase for STI was 1303.8(BAEE)/mL and 94.34(BAEE)/min/mL respectively.The half\|life of the immobilized proteinase was about 12 days at
文摘以4个抗豆象绿豆品系B18、B20、B24和A22为试材,以感虫绿豆品种晋绿1号为对照,研究了不同绿豆中胰蛋白酶抑制剂活性及其在高温、酸碱及超声波下绿豆的胰蛋白酶抑制剂稳定性。结果表明,4个抗豆象绿豆品种胰蛋白酶抑制剂活性均显著高于对照感虫品种,且均与对照在0.01水平差异极显著,其中B18活性最高,高达70.2TI U g–1,B20和A22活性次之,B24活性最差,但仍高达55.2 TI U g–1。4个抗豆象绿豆品种在不同温度、不同p H和不同振幅超声波下,残余活性均比对照高,且残余活性均随温度升高、温浴时间延长而降低,p H在2~12之间,随p H值的升高,残余活性均呈现先升高后降低的趋势,且p H值为6~8之间残余活性最高,残余活性也随超声波辐射强度升高、时间延长而降低,且4个抗虫品种中B18的耐高温性、耐酸碱性和耐辐射性最强,B20次之,B24的耐高温性、耐酸碱性最差,A22耐辐射性最差,说明在不同温度、p H和超声波处理后,B18、B20是抗豆象绿豆胰蛋白酶抑制剂残余活性保存最高的2个品种,应用价值较大。
