水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

地衣芽孢杆菌β—甘露聚糖酶的纯化及其性质的研究

地衣芽孢杆菌１Ｂａｃｉｕｕｓ Ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＢＬ－３０６产生的胞外β－甘露聚糖酶经硫酸铵分级盐析,ＤＥＡＥ－纤维素柱层析。Ｓｅｐｈａｄｅｘ－Ｇ１００柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化,得到ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）均一样品。用ＳＤＳ－ＰＡＧＥ测得纯化后β－甘露聚糖酶分子量为２６０００道尔顿。用凝胶等电聚焦电泳（ＰＡＧＥＩＥＦ）测得等电点ＰＩ为５．０。该酶     

枸杞子糖蛋白的分离纯化,物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析的步骤,从湖南产尖吻蝮蛇毒中纯化出两个出血毒素。ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸分别占２３％和２４％。ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具     

蕃茄蛋白酶抑制剂Ⅱa,Ⅱb的分离纯化及性质研究

经硫酸铵分级、ＣＭ－纤维素柱层析和Ｓｅｐｈａｄｅｘ Ｇ－７５柱层析,从野生型秘鲁蕃茄未成熟果实的匀浆液中分离纯化了蛋白酶抑制剂Ⅱａ,Ⅱｂ。纯化的蛋白酶抑制剂经ＳＤＳ－ＰＡＧＥ鉴定呈单一带,分子量均为１７ｋＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ结果表明蛋白酶抑制剂Ⅱａ,Ⅱｂ均与马铃薯蛋白酶抑制Ⅱ有血清学上的交叉反应。抑制蛋白酶活性测定显示两种蛋白酶抑制剂都表现强的抑制胰凝乳蛋白酶的活性,但对胰蛋白酶的     

枸杞子糖蛋白的分离纯化、物化性质及糖肽键特征

从宁夏枸杞子提取得到的粗多糖,经ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ和ＳｅｐｈａｄｅｘＧ－１００柱层析,得到均一的枸杞子糖蛋白ＬｂＧＰ。分子量由ＳＤＳ－ＰＡＧＥ测定为８８ｋｄ,糖含量为７０％,糖组成为Ａｒａ：Ｇａｌ：Ｇｌｃ＝２．５：１．０：１．０（摩尔比）,并含有其他１８种天然氨基酸。初步分析表明ＬｂＧＰ是Ｏ－连接的糖蛋白。     

高压快速柱分离纯化牛血球超氧化物歧化酶的研究

以牛血球为材料,经溶血等处理和丙酮沉淀,获得牛血球超氧化物歧化酶粗品。此粗酶可以通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＣＭ－Ｓｅｐｈａｒｏｓｅ快速柱层析,获得超氧化物歧化酶纯品。纯化的酶比活可达１３５００ｕ／ｍｇ,经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和快速蛋白液相色谱（ＦＰＬＣ）检测,结果表明,纯化酶是均一的Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤测得该酶分子量为３１,８００,ＳＤＳ－ＰＡＧＥ测得亚基分子量为１５     

水稻疏基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基因,水稻巯基蛋白酶抑制剂     

与光系统Ⅱ颗粒结合的对DTT敏感的蛋白酶的纯化和性质

菠菜光系统Ⅱ颗粒的１ｍｏｌ／ＬＮａＣｌ抽提液经ｂｕｔｙｌ－Ｔｏｙｏｐｅａｒｌ６５０Ｍ疏水层析和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析分离得一种对ＤＴＴ敏感的蛋白酶。用ＳＤＳ－ＰＡＧＥ和Ｓｕｐｅｒｏｓｅ１２凝胶过滤测得该酶的分子量为３７ｋＤ,其为单体酶。该酶可降解１８ｋＤ和２４ｋＤ水溶性蛋白质,分别产生１３．２ｋＤ、１２ｋＤ、１０．５ｋＤ和２３ｋＤ、２２ｋＤ、２０ｋＤ片段,最适ｐＨ为８．０,对ＮａＣｌ不敏感,对ＤＴＴ和β－Ｍｅ敏感。抑制实验表明该酶不属丝氨酸类蛋白酶。     

广东眼镜王蛇毒酸性磷脂酶A2的分离纯化及抗血小板聚集作用

目的 研究眼镜王蛇毒中酸性磷酸脂酶Ａ２对血小板的作用。方法 采用ＣＭ－Ｓｅｐｈａｄｅｓｘ Ｃ－２５Ｓｅｐｈａｄｅｘ Ｇ－７５,ＤＥＡＥ－Ｓｅｐ「ｈａｄｅｘＡ－２５,Ｓｅｐｈａｄｅｘ Ｇ－７５多步柱层析法,聚丙烯酰胺产胶电泳,酸性磷脂酶Ａ２酶活性测定;血小板聚集实验。累进要从粗中分离纯化出一酸性磷脂酶Ａ２单体,分子量为１４ＫＤ;等电点ＰＩ３．８;ＰＬＡ２比值２０μｍｏｌ．ｍｇ＾－１．ｍｉｎ＾－１;小     

A new type of low-molecular mass cysteine proteinase inhibitor from pig leukocytes

A new low-molecular mass cysteine proteinase inhibitor (CPI) was purified from the cytosol of peripheral pig leukocytes. The isolation procedure included DEAE chromatography, Sephadex G-100 gel filtration and fast-protein liquid chromatography on Mono Q. The procedure resulted in the isolation of a homogeneous protein with a molecular mass of approximately 12 kDa and a pI of 4.8. The amino terminus is blocked. The amino-acid composition and the sequence of the C-terminal part of the molecule are suggestive of a new family of cystatins. The CPI was found to be a tight-binding inhibitor of both papain and cathepsin L, with Ki values of 0.1 nM and 1 nM, respectively.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.     

A Proteinase from Mung Bean Sprouts That Inactivaties Soybean Trypsin Inhibitor (in English)

By 30%-60% (NH4)2SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (ProteinPM-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean (Vigna rabiata (L.) Wilczek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its Km and Vmax for STI were 769.2N-α-benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE·mL-1·min-1 respectively. This proteinase was stable at temperatures lower than 50℃ and pH 6.5-8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50℃ and pH 8.0 in 4 h.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

Purification and characterization of inhibitor against the cysteine proteinase of Rana catesbeiana

1. Inhibitors of cysteine proteinase were found in tadpole tail of metamorphosing bullfrog. 2. One of the inhibitors was purified by affinity chromatography with CM-papain agarose, gel filtration with Superose 12 and ion exchange chromatography with Mono S. 3. The molecular weight of the inhibitor was 130,000-140,000 and the isoelectric point was pH 9.6. 4. The inhibitor had inhibitory effects on ficin, papain and tadpole tail cysteine proteinase. 5. The inhibitor is possibly involved in the regulation of muscle degradation in tail regression of metamorphosing tadpole.     

Isolation, characterization and kinetics of goat cystatins

Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3–10 and up to 95 °C. Immunological identity with the sheep LMW kininogen was obtained suggesting that the inhibitor is closely related to kininogens. Spectral studies confirm that the inhibitors have predominantly an α-helical structure and undergo major conformational changes during complex formation with papain. The inhibitors had similar inhibitory activities on cysteine proteinases. Both inhibitors inhibited papain, ficin and bromelain competitively, with maximum affinity for papain. The overall lower affinity of these inhibitors to cysteine proteinases compared to other known cystatins can be attributed to the unusual N-terminal sequence where Leu is substituted by Ile. Furthermore, N-terminal sequence analysis revealed maximum homology to mammalian LMW kininogen.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10^{? 9} M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.     

水稻巯基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂（ＣＰＩ）经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达７２μｇ时,即可产生明显的抑制作用。