亲和素结合蛋白性质的研究

前曾发现在日本血吸虫虫卵中,存在一种新的能与亲和素专一性结合的蛋白质,称为亲和素结合蛋白,在分离纯化ＡＢＰ的基础上,用ＳＤＳ－ＰＡＧＥ及ｅｐｈａｄｅｘ Ｇ－１５０分别测定了ＡＢＰ的分子量,并做了糖蛋白染色及等电点测定,实验结果表明ＡＢＰ是一个分子量为６５ｋＤ的碱性糖蛋白,由数个相同的分子量为１２．７ｋＤ的亚单位组成。ＳＤＳ、β－硫基乙醇处理的ＤＤＢＰ仍能与亲和素结合,提示ＡＢＰ的亚单位能与亲和素结     

抗癌胚抗原单链抗体基因与核心链霉亲和素基因融 …

将抗癌胚抗原单链抗体基因与核心链霉亲和素基因融合插入昆虫杆状病毒供体质粒ｐＦａｓｔＢａｃＨＴａ中,在粉纹夜蛾Ｔｎ－５Ｂ１－４细胞中进行表达。ＳＤＳ－ＰＡＧＥ分析结果表明,表达产物分子量为４１ｋＤ左右,Ｗｅｓｔｅｒｎ印迹分析结果表明,以ＨＲＰ标记的生物素进行蛋白质印迹在４１ｋＤ处可见表达条带,表明融合蛋白能特异性的与生物素结合,放射免疫分析表明重组杆状病毒表达产生的ＳｃＦｖ－ＣＳ蛋白能特异性结合癌胚     

人肝癌细胞的IGF—BP1分子特性及内源性蛋白酶对其降解的研究

用ＳＤＳ－ＰＡＧＥ电泳、高效液相色谱（ＨＰＬＣ）、质谱等方法,研究了人肝癌细胞（ＨｅｐＧ２）分泌的胰岛素样生长因子结合蛋白－１（＾３５Ｓ－ＩＧＦ－ＢＰ１）的分子结构、特性,及其被内源性蛋白酶降解的特点,＾３５Ｓ－ＩＧＦ－ＢＰ１的经抗体免疫沉淀、生化分离,纯化为均一体,其分子是由多个亚构成的蛋白质,分子量约为２７ｋＤ;细胞ＵＭＲ、ＨｅｐＧ２、Ｈ３５ＢＲＬ３Ａ分泌的蛋白酶能将＾３５Ｓ－ＩＧＦ－ＢＰ１催     

菜豆下胚轴中的细胞分裂素结合蛋白

用植物体内天然存在并具有很高活性的玉米素（ｔ－Ｚ）和异戊烯基腺苷（ｉＰＡ）作为配基,分别与Ｓｅｐｈａｒｏｓｅ－４Ｂ偶联,制成亲和吸附介质,分离、纯化菜豆（Ｐｈａｓｅｏｌｕｓ ｖｕｌｇａｒｉｓ）黄化幼苗下胚轴中的细胞分裂素结合蛋白。一种分子量为１５．５ ｋＤ（简称ＺＢＰ）,只含一个多肽链;另一种分子量为１６５ ｋＤ（简称ＩＢＰ）,含有两种亚基,分子量分别为４３ ｋＤ和４０ ｋＤ。对ＺＢＰ的结合活性进行了研究,发现ＺＢＰ与ｔ－Ｚ结合时的解离常数（Ｋｄ）为３．２×１０－ ７ ｍ ｏｌ／Ｌ。经计算,每个ＺＢＰ分子只有一个ｔ－Ｚ结合位点     

人早期胎盘绒毛纤维连接蛋白的分离纯化及鉴定

人妊娠５－８周的胎盘绒毛经匀浆后,用２ｍｏｌ／Ｌ ｕｒｅａ－ＰＢＳ提取,通过Ｈｅｐａｒｉｎ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和柱层析,再经Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ凝胶过滤层析,得到人早期胎盘纤维连接蛋白（ｅａｒｌｙ ｐｌａｃｅｎｔａ ｆｉｂ－ｂｒｏｎｅｃｔｉｎ,ｅｐＦＮ）。经还原及非还原ＳＤＳ－ＰＡＧＥ和免疫印迹电泳分析,ｅｐＦＮ分子量约５００ｋＤ,是由两个２５０ｋＤ亚基组成,与人足月胎盘纤维连接     

红苋P104种子谷蛋白的电泳分析

通过单向不平变性聚丙烯酰胺凝胶等电聚焦（ＩＥＦ）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）和双向电泳（ＩＥＦ×ＳＤＳ－ＰＡＧＥ）分析了红苋Ｒ１０４种子谷蛋白的亚基组成,亚其分子量和等电点分布。谷蛋白的双向电泳图谱可分辨出１００多个亚基成分,其主要亚基为：５４ｋＤ（ｐＩ７．１５）;３３ｋＤ（ｐＩ５．８２）;３１ｋＤ（ｐＩ６．９２;ｐＩ６．７０;ｐＩ６．６５）;２２ｋＤ（ｐＩ８．３４）;２     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

高等植物的泛肽素基因及其基因表达

高等植物的泛肽素基因及其基因表达印莉萍,刘兴军,林忠平（首都师范大学生物系北京１０００３７）（中科院植物所北京１０００４４）ＵＢＩＯＵＩＴＩＮＧＥＮＥＡＨＤＪＤＳＧＥＮＥＥＸＰＲＥＳＳＩＯＮＩＮＨＩＧＨＥＲＰＬＡＮＴＳ￥ＹｉｎＬｉ－ｐｉｎｇ;ＬｉｕＸ...     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

巨尾阿丽蝇幼虫肠道蛋白酶的分离纯化及性质

巨尾阿丽蝇幼虫肠液ＳＤＳ－ＰＡＧＥ后,Ｘ光片显影呈现３条蛋白酶活性带。ＩＥＦ后出现２条蛋白酶活性带,等电点分别为ＰＨ８．５和ＰＨ７．７。肠液经硫铵沉淀,ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＳｅｐｈａｄｅｘＤＥＡＥＡ－２５离子交换和ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,分离化出分子量约为１４ＫＤ的巨尾阿丽蝇蛋白酶。     

虎纹捕鸟蛛毒液中透明质酸酶的纯化和部分性质的研究

用分子筛（岛津ＤＩＯＬ－１５０柱）和阳离子交换（岛津ＷＣＸ－１柱）高效液相色谱从虎纹捕鸟蛛（Ｓｅｌｅｎｏｃｏｓｍｉａｈｕｗｅｎａ）毒液中分离提纯透明质酸酶（Ｈｙａｌｕｒｏｎｉｄａｓｅ,ＥＣ３．２．１．３５）．经等电聚焦电泳为一条带,ｐＩ＝７．２．经ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为４０ｋＤ,经凝胶过滤测得分子量为４０．７ｋＤ。以透明质酸为底物时在ｐＨ３．５─５．５范围内有较大活性,最适ｐＨ值为４．０;在ｐＨ４．５─６．０范围内稳定,在反应温度为３０─６０℃时有较大活性,最适温度为５０℃;对热稳定,０．１５ｍｏｌ／Ｌ的ＮａＣｌ对酶活性有一定的稳定作用．３％的肝素、５００μｍｏｌ／Ｌ的Ｈｇ~（２＋）、Ｆｅ~（２＋）、Ｃｕ~（２＋）对酶活性有明显的抑制作用。     

抗生物素蛋白结合蛋白的发现及分离纯化

本文报道了在日本血吸虫感染的兔血清及日本血吸虫卵中,存在一种能与抗生物素蛋白（又称亲和素）专一性结合的蛋白质,称为抗生物素蛋白结合蛋白。该蛋白质与亲和素的结合与生物素及亲和素的糖链部分无关,并能在高ＰＨ、高浓度盐及去污剂等条件下与亲和素结合。利用ＤＥＡ－５２离子交换柱及偶联有亲和素的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱,从日本血吸虫感染的兔血清中,分离纯化了该蛋白质。提纯的亲和素结合蛋白在ＳＤＳ－ＰＡ     

SeO_3~（2－）及SeO_3~（2－）和半胱氨酸或还原型谷胱甘肽联用对牛脑V

Ｓｅ_３~（２－）对牛脑Ｖ型质子转运ＡＴＰ酶复合体活力呈现明显的抑制作用。这种抑制作用具有对剂量和预保温时间的依赖关系,且不被稀释效应所恢复,但过量的ＤＴＴ可去除这种抑制作用。半胱氨酸对这种抑制作用呈双功能效应。低浓度时促进抑制作用,高浓度时部分恢复抑制作用。还原型谷胱甘肽与半胱氨酸有类似效应。但促进抑制作用没有半胱氨酸强,而恢复抑制作用却较半胱氨酸强。ＳＤＳ聚丙烯酰胺凝胶电泳和放射自显影图谱表明,ＳｅＯ_３~（２－）和半脱氨酸优先结合在７０ｋＤ亚基上。并且验证了ＡＴＰ酶复合体的－ＳＨ基和ＳｅＯ_３~（２－）及半胱氨酸的－ＳＨ基以－Ｓ－Ｓｅ－Ｓ－键相连接。根据上述结果,讨论了７０ｋＤ亚基的－ＳＨ基及其相关的结构域涉及到ＡＴＰ酶的活性中心,７０ｋＤ亚基可能是ＡＴＰ酶复合体的水解活性亚基。     

胞外青霉素酰化酶的纯化及部分理化性质

巨大芽孢杆菌产胞外青霉素酰化酶发酵液经硫酸铵分级抽提及ＳｅｐｈａｄｅｘＧ－１００、羟基磷灰石、ＤＥＡＥ纤维素ＤＥ５２等层析步骤,提纯了青霉素酰化酶,得到电泳均一的酶制剂。纯酶比活力约为２５Ｕ／ｍｇ蛋白,纯化４９倍,活力回收５８％,经ＰＡＧＥ及ＳＤＳ－ＰＡＧＥ测知该酶不含亚基,其分子量约为１４０ｋＤ。该酶最适ｐＨ为９．０,最适温度４７℃,用底物ＮＩＰＡＢ测活,其Ｋｍ值为６．２×１０~（－４）ｍｏｌ／Ｌ,Ｖｍ值为１．２４×１０４ｍｏｌ／Ｌ。此外还探讨了部分金属离子对该酶的影响。     

Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin filaments. The large size of the leaves, their interruption by two hinges and flexible actin-binding site, facilitate cross-linking of widely dispersed actin filaments.     

中药固真方对促甲状腺素受体基因表达的调节作用

促甲状腺素（ＴＳＨ）通过甲状腺细胞膜上的ＴＳＨ受体（ＴＳＨＲ）,产生第二信使ｃＡＭＰ,从而激活ｃＡＭＰ反应性起动子,而使相应的基因获得表达．实验结果表明,在构建有ＴＳＨＲ和糖蛋白激素ｃＡＭＰ反应性起动子以及萤光素基因（Ｌｕｃ）的转染细胞中,经补肾益精中药固真方提取液（１×１０~（－４）稀释）处理３～５ｄ后,可下调ＴＳＨＲ基因的表达,并使ＴＳＨＲ数目减少．提示固真方可调整甲状腺细胞的功能,或许有利于调整甲状腺机能亢进．     

A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase.

We have used the photoaffinity label azido-[3H]IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analog of indole-3-acetic acid, to identify auxin-binding proteins (ABPs) in the soluble fraction of Hyoscyamus muticus. A 25-kD polypeptide previously described (H. Macdonald, A. M. Jones, P. J. King [1991] J Biol Chem 266: 7393-7399) has now been purified to homogeneity by conventional methods. Binding of azido-[3H]IAA to the purified protein was reduced by active auxins but not by inactive indoles. Partial amino acid sequences of the purified protein showed high homology to glutathione S-transferase (GST) from tobacco (ParB) and from maize (GT32). The conclusion that the 25-kD ABP is a GST is further supported by high GST activity in fractions highly enriched in the 25-kD polypeptide and recognition of the ABP by antibodies against GST from wheat and maize. Furthermore, purification of a protein from a soluble protein extract from H. muticus by affinity chromatography on glutathione-agarose also yielded a 25-kD polypeptide that was indistinguishable in its N-terminal amino acid sequence and biochemical characteristics from the protein purified by conventional methods. Possible functions of GST in auxin action are discussed.     

蜚蠊灭菌肽的诱导及初步分离分析

昆虫经诱导盾产生灭菌肽的研究近年来已有很大进展,有关这方面的研究工作绝大多数都是以有翅亚纲内生翅类鳞翅目(主要是蚕类)昆虫和少数双翅目昆虫为材料.本文首次以有翅亚纲外生翅类蜚蠊目的美洲蜚蠊(Ptriplaneta americana L.)为实验昆虫,用Escherichia coil K₁₂ strain D₃₁作诱导源,对不同发育期、不同性别、不同成虫期的蜚蠊进行诱导后,采用含菌培养基平板测活方法,就存在个体数及能产生抗菌物质的个体数进行了初步的研究.发现成虫日龄在10天之内的雄性蜚蠊能够产生抗菌物质的个体百分比最高.抗菌物质出现的高峰期是在诱导后第三、四天.用滴滴涕和溴氰菊酯作诱导源对雄性蜚蠊的诱导实验表明,杀虫剂也能诱导蜚蠊产生抗菌物质,而且所诱导产生抗菌物质的活性强度(用抑菌圈直径表示)高于大肠杆菌所诱导的.滴滴涕和溴氰菊酯的重复诱导可提高蜚蠊产生抗菌物质个体百分比.蜚蠊经诱导后产生的抗菌物质具有广谱性,对苏云金杆菌、金黄色葡萄球菌、枯草杆菌及绿脓杆菌等有较强的抗菌活性,而对大肠杆菌D₃₁、大肠杆菌、粘质沙雷氏杆菌和溶壁微球菌等有较弱的抗菌活性.用肽类物质的指纹图谱法分离蜚蠊血淋巴抗菌物质,发现经诱导后血淋巴中确有新的肽类物质产生,该物质具抗菌活性,用DABITC法分析,其N-末端氨基酸为赖氨酸.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Vitamin E protects nerve cells from amyloid beta protein toxicity.

The amyloid beta protein (ABP) is a 40 to 42 amino acid peptide which accumulates in Alzheimer's disease plaques. It has been demonstrated that this peptide and a fragment derived from it are cytotoxic for cultured cortical nerve cells. It is shown here that ABP and an internal fragment encompassing residues 25 to 35 (beta 25-35) are cytotoxic to a clone of PC12 cells at concentrations above 1 x 10(-9)M and to several other cell lines at higher concentrations. Between 10(-9) and 10(-11) M beta 25-35 protects PC12 cells from glutamate toxicity. The antioxidant and free radical scavenger vitamin E inhibits ABP induced cell death. These results have implications regarding the prevention and treatment of Alzheimer's disease.