分离和鉴定沙冬青抗冻蛋白质

采用经典的蛋白质色谱技术,纯化沙冬青（ Ａｍｍｏｐｉｐｔａｎｔｈｕｓ ｍｏｎｇｏｌｉｃｕｓ （Ｍａｘｉｍ ．） Ｃｈｅｎｇｆ．） 叶片中的抗冻蛋白质,然后经非变性ＰＡＧＥ胶分离、回收,得到具有热滞活性的两条带：Ｂ１ 和Ｂ３。前者在８ ｇ／Ｌ时热滞活性为０．４６ ℃,并在ＳＤＳ＿ＰＡＧＥ胶上分离出两条带（ 分子量为６７ ｋＤ和２１ ｋＤ） ;后者在１０ ｇ／Ｌ时热滞活性为０．４５ ℃,在ＳＤＳ＿ＰＡＧＥ胶上只有１ 条带（ 分子量为３９．８ ｋＤ）。Ｂ１ 和Ｂ３ 均不能被Ｓｈｉｆｆ 试剂染色,也不具有典型糖蛋白的紫外吸收特征,因而可能不是糖蛋白     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值,在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）;在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ）,４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４）,表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。     

枯草芽孢杆菌中性β-甘露聚糖酶的纯化及性质研究

经硫酸铵分级沉淀、超滤浓缩、阴离子交换层析和凝胶过滤层析,由枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）ＢＭ９６０２培养滤液得到了常规凝胶电泳一条带的中性β－甘露聚糖酶．该酶具有与其它已知同类酶相类似的性质,但用ＳＤＳ－ＰＡＧＥ测得该酶分子量为３７ｋＤ;用聚丙烯酰胺等电聚焦电泳测得等电点ｐＩ为４．９．     

大熊猫乳酸脱氢酶同工酶H_4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５＇－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出了乳酸脱氢酶同工酶Ｈ４．纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＰＡＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分子量为３６０００,等电点为５．４５．经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ．氨基酸组成分析表明每个亚基含有５个Ｃｙｓ,９个Ｍｅｔ．     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ     

从感染TMV的番茄叶纯化胞外β—N—乙酰氨基已糖苷酶

番茄系统感染ＴＭＶ（ｔｏｂａｃｃｏｍｏｓａｉｃｖｉｒｕｓ）诱导叶β－Ｎ－乙酰氨基已糖苷酶活性升高。番茄叶胞外提取液经冰冻干燥浓缩,－２０℃丙酮沉淀、ＣＭ－ＳｅｐｈａｄｅｃＣ－２５离子交的层析,ＰＢＥ９４（ＰｏｌｙｂｕｆｆｅｒＥｘｃｈａｎｇｅｒ９４,Ｓｉｇｍａ）聚焦层析和ＳＥＰＨＡＤＥＸｇ－１５０凝胶层析纯化,获得纯化的β－Ｎ－乙酰氨基乙糖苷酶。凝胶层析测得该酶的分子量为１４５ｋＤ,ＳＤＳ－ＰＡＧＥ     

盐生杜氏藻生油—3—磷酸脱氢酶的分离化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,Ｂｈｕｅ Ｓｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化直二氏藻甘油三磷酸（Ｇ－３－Ｐ）脱氢酶（ＥＣ１．１．１．８）得到比活为１２．６ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４－２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳ－ＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶     

柑叶片蔗糖酶的分离纯化及其部分性质的研究

柑（Ｃｉｔｒｕｓｒｅｔｉｃｕｌａｔａ Ｂｌａｎｃｏ）幼叶中存在高活性的酸性蔗糖酶。经硫酸铵盐析、ＤＥＡＥ－琼脂糖离子交换层析、ＳｅｐｈａｃｒｙｌＳ－２００ 凝胶层析纯化,活性回收率６．４％ ,纯化倍数１７９．２ 倍。纯化的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,ＳＤＳ－ＰＡＧＥ显示１ 条蛋白带,其亚基分子量４０ ｋＤ。用ＳｅｐｈａｃｒｙｌＳ－２００ 凝胶层析法测得分子量为８０ ｋＤ。推测该酶由两个相同亚基构成。以蔗糖为底物测定该酶的表观Ｋｍ 为１．６×１０－ ２ ｍ ｏｌ·Ｌ－ １,Ｖｍ ａｘ为１００ ｍ ｇ 还原糖·ｍ ｇ－ １蛋白质·ｈ－ １。最适ｐＨ ５．０,酸碱稳定区在ｐＨ ４．５—５．５ 之间。最适温度５５℃     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

圆弧青霉碱性脂肪酶的分离纯化的特性

圆弧青霉突变株ＰＧ３７发酵液经离心、硫酸铵盐析、疏水层析、阴离子交换层析和凝胶过滤分离纯化得到了比活性为每毫克蛋白质５２００ｕ的碱性脂肪酶,纯化倍数１６．５,得率３３．２％,在聚丙烯酰胺凝胶电泳（ＰＡＧＥ）和ＳＤＳ－聚丙烯酰胺凝胶电脉（ＳＤＳ－ＰＡＧＥ）上均呈现单一 白质条带。ＳＤＳ－ＰＡＧＥ和凝胶过滤分别测得酶的分子量为２７．５ｋＤ和２９．ｋＤ,表明该酶以单体形式存在。Ｎ末端１０个氨基酸的序列测     

Glutathione S-transferase isoenzymes in the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae)

Isoenzymes of glutathione S-transferase (GST) in adult Adalia bipunctata, an aphidophagous predator, were studied. Cytosolic GST activity was studied in each beetle developmental stage. The highest activities towards both 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dinitro-1-iodobenzene (DNIB) occurred in adults. The enzyme distribution was investigated in adults. While most of the enzymatic activity was found in the abdomen (40-50 and 34-63% respectively) using several concentrations of both CDNB and DNIB, significant differences were observed for the head and the thorax depending on the substrate. Activities were more abundant in the thorax with DNIB (37-47%) compared to the 13-19% obtained with CDNB. Some GST activity was also detected in the elytra. GSTs were purified by epoxy-activated Sepharose 6B affinity chromatography and applied to an HPLC column to determine the native molecular weight (69 kDa). Three isoenzymes were separated by chromatofocusing at pH ranges 7-4. Three bands with molecular mass from 23 to 26 kDa were visualised on SDS-PAGE. Their isoelectric points were 6.66, 6.36, and 6.21. The substrate specificities and the kinetic parameters (Vm and Km) of the isoenzymes showed large differences depending on the isoenzyme. Arch.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

Characterization of glutathione transferase from Gammarus italicus.

1. By using affinity chromatography and chromatofocusing analysis at least two major glutathione transferases, named GST II and GST III can be isolated from Gammarus italicus. 2. GST II has an isoelectric point at pH 5.0 and is composed of two subunits with an apparent molecular mass of 28 KDa. 3. GST III which has an isoelectric point at pH 4.6 was found to be an heterodimer of 27 KDa and 28 KDa. 4. The 28 KDa subunit cross-reacted in immunoblotting analysis with antisera raised against pi class GST, whereas none of the antisera raised against alpha, mu and pi class GSTs cross-reacted with the 27 KDa subunit.     

Glutathione transferases in the tapeworm Moniezia expansa.

Four forms of GSH transferase were resolved from Moniezia expansa cytosol by GSH-Sepharose affinity chromatography and chromatofocusing in the range pH 6-4, and the presence of isoenzymes was further suggested by analytical isoelectric focusing. The four GSH transferase forms in the cestode showed no clear biochemical relationship to any one mammalian GSH transferase family. The N-terminal of the major GSH transferase form showed sequence homology with the Mu and Alpha family GSH transferases. The major GSH transferase appeared to bind a number of commercially available anthelmintics but did not appear to conjugate the compounds with GSH. The major GSH transferase efficiently conjugated members of the trans-alk-2-enal and trans,trans-alka-2,4-dienal series, established secondary products of lipid peroxidation.     

Purification and characterization of acidic form of glutathione S-transferase in human fetal livers: high similarity to placental form.

An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver

Cytosolic GSH transferases have been purified from rat lung by affinity chromatography followed by chromatofocusing. On the criteria of order of elution, substrate specificity, apparent subunit Mr, sensitivity to inhibitors, and reaction with antibodies, transferase subunits equivalent to subunits 2, 3, and 4, in the binary combinations occurring in liver, were identified. However, subunit 1 (and therefore transferases 1-1 and 1-2) was not detected. The most conspicuous difference is the presence in lung of a new form, eluting at pH 8.7, which is not detected in rat liver. This isoenzyme (transferase "pH 8.7") is characterized by its low apparent subunit Mr and high efficiency in the conjugation of glutathione with anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, considered the ultimate carcinogen of benzo(a)-pyrene.     

人胎肺细胞的条件化培养液中两种类型纤溶酶原活化物的分离

用正常人胎肺细胞体外培养,从其培养液中分离纤溶酶原活化物（ＰＡ）,通过ＣＭ－ＳｅｐｈａｄｅｘＣ－５０层析,硫酸铵沉淀和Ｆｉｂｒｉｎ－Ｓｅｐｈａｒｏｓｅ,Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ亲和层析及ＳｅｐｈａｄｅｘＧ－５０凝胶过滤等步骤,从１０．５ｌ条件培养液中分离纯化得到两种类型的纤溶酶原活化物,ｔ－ＰＡ９０μｇ,ｕ－ＰＡ８００μｇ．在还原条件下ＳＤＳ－ＰＡＧＥ均显示单带,分子量ｔ－ＰＡ为７２ｋＤ,ｕ－ＰＡ为５４ｋＤ,纤溶比活分别为１５６０００ＩＵ／ｍｇ蛋白和１０６０００ＩＵ／ｍｇ蛋白．