条斑紫菜蛋白酶解产物中α-葡萄糖苷酶抑制剂纯化及特性

以α-葡萄糖苷酶抑制活性为指标,优选出1398中性蛋白酶、碱性蛋白酶和氨肽酶在一定条件下复配酶解条斑紫菜蛋白制备α-葡萄糖苷酶抑制剂。酶解液加入乙醇至终浓度为60%以沉淀去除多糖,上清液为α-葡萄糖苷酶抑制剂粗品。该粗品利用SP Sepharose High Performance阳离子交换层析、Sephadex G-10凝胶层析和Mono Q阴离子交换层析进行分离纯化,获得一种肽类α-葡萄糖苷酶抑制剂(LGI)。LGI经反向高效液相层析测定纯度为62.4%,基本特性分析显示其具较好的温度和pH稳定性,对α-葡萄糖苷酶半抑制浓度IC50值为97.36μg/mL,属于一种非竞争性抑制剂。     

一种云芝发酵所产胃蛋白酶抑制剂的纯化及部分性质研究

云芝发酵液经超声波细胞破碎,离心和大孔树脂脱色得上清液.上清液再经DEAE-52、Mono Q离子交换层析和UltroGel ACA-54凝胶过滤分离得到一种胃蛋白酶抑制剂.它的成分是蛋白质,经UltroGel ACA-54凝胶过滤和SDS-PAGE鉴定为单亚基,相对分子量为22.31 kDa.该抑制剂热稳定性良好,对胃酶有很强的抑制作用,半抑制浓度IC50值为29.26 μg/mL,抑制类型属于非竞争和反竞争混合型抑制模式,其Ki值为0.996 μmol/L.     

螺旋藻源血管紧张素转化酶抑制肽的纯化和鉴定

血管紧张素转化酶(ACE)抑制剂通过影响肾素-血管紧张素系统,对减缓和抑制高血压具有重要的作用.该研究通过超滤、凝胶过滤色谱、反相高效液相色谱等方法,从钝顶螺旋藻的木瓜蛋白酶水解液中分离、纯化得到一种血管紧张素转化酶(ACE)抑制肽,并利用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)和氨基酸测序对纯化肽进行鉴定.此外,对其抑制类型和体外模拟消化环境稳定性也进行了研究.结果表明,分子质量范围为0～3000ku的酶解液ACE抑制活性最高,IC50值为(1.03±0.04)g/L.该部分酶解液通过纯化获得ACE抑制肽,IC50值为(0.0094±0.0002)g/L,相当于(27.36±0.14)μmol/L,序列经鉴定为Val-Glu-Pro.Lineweaver-Burk图和Dixon图表明该ACE抑制肽为非竞争性抑制剂,Ki值为(23.59±0.54)μmol/L.体外稳定性实验显示,该抑制肽在胃蛋白酶、胰凝乳蛋白酶、胰蛋白酶等胃肠蛋白酶的消化下能够保持良好的抑制活性,表明螺旋藻源ACE抑制肽可以用于降血压功能食品和药剂方面,具有很好的发展前景.     

栗精碱对棉铃虫中肠组织可溶型海藻糖酶的抑制效应

【目的】研究一种植物源化合物栗精碱对棉铃虫Helicoverpa armigera(Hübner)5龄幼虫中肠组织可溶型海藻糖酶的活体和离体抑制效果,初步探索其抑制作用。【方法】经过离子交换层析和疏水层析从棉铃虫5龄幼虫中肠组织分离纯化得到可溶型海藻糖酶,通过凝胶过滤层析确定其分子量。分别用不同浓度的抑制剂进行离体和活体抑制研究,得出栗精碱对可溶型海藻糖酶的抑制率和抑制中浓度(IC50),并采用Lineweaver-Burk和Dixon作图法分析抑制类型。【结果】棉铃虫中肠可溶型海藻糖酶比活力为2.022 U/mg,回收率为67.57%,纯化倍数为23.84,分子量约为67 k Da。离体抑制研究表明,3.75,7.5,15和30μmol/L的栗精碱对可溶型海藻糖酶的抑制率分别为42.00%,50.30%,58.32%和67.33%;抑制中浓度(IC50)为7.32μmol/L。通过LineweaverBurk和Dixon作图法,确定栗精碱是棉铃虫中肠组织可溶型海藻糖酶的一种有效的竞争性抑制剂,Ki值为4.9μmol/L。活体抑制研究表明,分别注射浓度为30,15和7.5μmol/L栗精碱10和20 h后,棉铃虫中肠可溶型海藻糖酶活性被显著抑制(P0.05)。相应地,注射后随着时间的延长,血淋巴海藻糖含量连续增加。而注射栗精碱20 h后,血淋巴葡萄糖浓度显著下降,使棉铃虫的能量供给出现障碍。【结论】结果表明,栗精碱是棉铃虫中肠可溶型海藻糖酶的一种有效的抑制剂,可为下一步开发有效的棉铃虫杀虫剂提供理论支持。     

萌发绿豆种子中的一种大豆胰蛋白酶抑制剂钝化酶

通过 30 %～ 6 0 % (NH4 ) 2 SO4 分级沉淀、DEAE_SepharoseCL_6B离子交换层析、SephacrylS_2 0 0凝胶过滤层析和WatersAP_1离子交换层析 ,从萌发的绿豆 (Vignarabiata (L .)Wilczek)种子中分离纯化出一种可降解大豆胰蛋白酶抑制剂 (STI)的蛋白酶。SDS_PAGE测定该酶的分子量为 2 9.8kD。该酶催化降解STI的Km 值为 76 9.2BAEE/mL ,Vmax为 115 .3BAEE·mL-1·min-1。该酶在 5 0℃、pH 8.0、相对酶活力 5 0 0 0BAEE/mL和 4h的反应时间时可将脱脂大豆粉中的STI活性钝化 90 .91%。该酶在温度低于 5 0℃及pH 6 .5～ 8.5时能保持其活性。     

尖镰孢青霉素V酰化酶的纯化及性质

通过硫酸铵沉淀、硅藻土吸附、DEAD-纤维素离子交换层析和Sephadex G-200凝胶过滤,由尖镰孢(Fusarium oxysporum)FP941培养滤液中得到了聚丙烯酰胺凝胶电泳均一的青霉素V酰化酶。酶作用最适pH为7.0,最适温度为50℃。酶在pH6.0—8.0和42℃以下稳定。酶作用青霉素V的米氏常数Km为4.65×10~(-3)mol/L;苯氧乙酸是酶的竞争性抑制剂,抑制常数Ki为23.87×10~(-3)mol/L;6-氨基青霉烷酸是酶的非竞争性抑制剂,抑制常数Ki为30.01×10~(-3)mol/L。某些金属离子对酶有抑制作用,Fe~(2+)最强,其次是Hg~(2+)和Cu~(2+)。用SDS凝胶电泳测定酶亚基分子量为77600;用分子筛测定自然酶分子量为148000。     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA 处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

云芝发酵产胃蛋白酶抑制剂的反应机理探讨

目的:对一种从云芝发酵液中提取得到的胃蛋白酶抑制剂的动力学性质和反应机理进行研究.方法:采用Lineweaver-Bunk双倒数法和UltroGelACA-54凝胶过滤法:结果:实验证明抑制剂为胞内产物.其成分是蛋白质,分子量为23 100Da,K值为0.996 μmol/L.结论:抑制剂对胃酶的抑制类型属于非竞争和反竞争混合型抑制模式,它与胃酶结合作用的方式可能是在酶的活性基同形成氢键并封锁酶与底物的结合部位.     

Purification and properties of an aspartic protease from potato tuber that is inhibited by a basic chitinase

A protease was isolated from potato ( Solanum tuberosum L. cv. Huinkul) tuber disks after 24 h of aeration when proteolysis is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration, it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it had a pH optimum between 4 and 5 and it was inhibited by pepstatin. Partial homology with other plant aspartic proteinases was observed in two sequenced tryptic fragments. It binds to Sepharose-concanavalin A and can be eluted with α -methyl mannoside, indicating that it is possibly glycosylated. Unlike other aspartic proteinases from Solanaceae that degrade pathogenesis-related proteins, it is unable to cleave a basic chitinase from potato. Moreover, this aspartic protease is strongly inhibited by the basic chitinase; the 50% inhibition is obtained when the molar ratio approaches 1, the same as with pepstatin. The interaction between this aspartic protease and a new type of endogenous inhibitor may be an interesting starting point to study the regulation of these aspartic proteases during stress.     

蚯蚓纤溶酶的糖成分分析

以大豆胰蛋白酶抑制剂为配基的亲和层析制备的蚯蚓纤溶酶是一组糖蛋白。通过苯酚一硫酸法测中性糖,3,5一二硝基水扬酸法测还原糖,硅胶G薄层层析法鉴定糖的种类,结果为：蚯蚓纤溶酶主要含有中性己糖,含量为15％左右,TFMS的去糖处理会使蚯蚓纤溶酶酶活丢失．     

pH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme

The redox enzyme violaxanthin de-epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE-I predominated at pH 7.2. On lowering the pH of the chromatography, VDE-I decreased and VDE-II increased. Furthermore, re-chromatography of either peak yielded both peaks. These results suggest that VDE-I and VDE-II are interconvertible depending on pH, and thus, they represent the de-protonated and protonated forms of the enzyme, respectively. Presumably the protonation-induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.     

Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

Direct Resolution of dl-Lysine-3, 5-Dimtrobenzoate

The inhibitory activity of an angiotensin I-converting enzyme (ACE) detected in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography on Bio-gel P-2 after treating with ethanol. The Hw fraction reduced the blood pressure in hypertensive rats after orally administering, while the Lw fraction did not. The ACE inhibitor in the Hw fraction was further purified by Dowex 50W ion-exchange chromatography and four subsequent steps of HPLC. On the basis of the SIMS-mass spectrum, NMR spectrum and other characteristics, the purified ACE inhibitor was identified as nicotianamine (N-[N-(3-amino-3-carboxypropyl)-3-amino-3- carboxypropyl]azetidine-2-carboxylic acid). The IC₅₀ value for this ACE was 0.26 µM.     

Purification and characterization of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol-treated rats

A low-molecular-weight protein, induced in rats following prolonged isoproterenol treatment, has been purified from rat submandibular glands by chromatography on columns of Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephadex G-75. The purified protein is homogeneous based on gel electrophoresis and Ouchterlony double diffusion. The molecular weight of the purified protein was 14,000 and 15,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Superose 12 column, respectively. This protein contains 31% glutamic acid/glutamine and aspartic acid/asparagine, 3.6% cysteine, and 2.5% proline. This protein is shown to be an inhibitor of several cysteine proteinases, papain and ficin being inhibited very strongly in approximately 1:1 molar ratio of enzyme to inhibitor. The protein is not detected in normal rat tissues but is induced in submandibular and sublingual glands even after 1 day of isoproterenol treatment of rats as early as 7 days after birth. Based on cysteine proteinase inhibitor activity, molecular size, and chemical composition this protein appears to belong to the cystatin superfamily.     

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([³⁵S]Met)preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-d -Leu-Pro-Phe-Val-d -Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys¹⁷²-Arg¹⁷³ pair located at the COOH-terminus of (Met)enkephalin-Arg⁶-Gly⁷-Leu⁸, as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze ³⁵S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Nature of the tryptic/chymotryptic inhibitor from horsegram (Dolichos biflorus).

A protease inhibitor specific to trypsin and chymotrypsin was purified from horsegram (Dolichos biflorus) with the inhibition index 0.24 micrograms/micrograms for trypsin and 0.36 micrograms/micrograms for chymotrypsin. In SDS-PAGE, the inhibitor protein was seen as a single band with apparent molecular mass Mr = 15,500. However, on fast protein liquid chromatography (FPLC) or non-denaturating PAGE, the inhibitor resolved into four components revealing the existence of isoinhibitors. Data on amino acid analysis indicate that the isoinhibitors are closely related. The major amino acids in the inhibitor are half cystine (18.9 mole %), aspartic acid (12.7 mole %) and serine (14.3 mole %). The inhibitor was partially stable to 0.1% sodium dodecyl sulphate, 8M urea or 6M guanidine hydrochloride. The inhibitory activity was lost on reduction or carboxamidomethylation or acetylation. Modification of the arginine groups or CNBr cleavage of the protein did not result in significant loss of either tryptic or chymotryptic inhibitory activities. The isoinhibitors separated by FPLC reacted with polyclonal antibody raised in rabbits and had pI values ranging from 4.8-5.1. The horsegram inhibitor thus resembles other Bowman-Birk protease inhibitors.     

Biochemical characterization of an aspartic protease from Vigna radiata: Kinetic interactions with the classical inhibitor pepstatin implicating a tight binding mechanism

Aspartic proteases are the focus of recent research interest in understanding the physiological importance of this class of enzymes in plants. This is the first report of an aspartic protease from the seeds of Vigna radiata. The aspartic protease was purified to homogeneity by fractional ammonium sulfate precipitation and pepstatin-A agarose affinity column. It was found to have a molecular weight of 67,406 Da by gel filtration chromatography. SDS-PAGE analysis revealed the presence of a heterodimer with subunits of molecular weights of 44,024 and 23,349 Da respectively. The enzyme was pH stable with the amino acid analysis confirming the molecular weight of the protein. The substrate cleavage site as analyzed by using the synthetic substrate was found to be the Phe-Tyr bond. The kinetic interactions of the enzyme were studied with the universal inhibitor, pepstatin A. This is the first report on the interactions of a plant aspartic protease with pepstatin-A, an inhibitor from a microbial source. A competitive one-step mechanism of binding is observed. The progress curves are time-dependent and consistent with tight binding inhibition. The K(i) value of the reversible complex of pepstatin with the enzyme was 0.87 microM whereas the overall inhibition constant K(i)* was 0.727 microM.