孔石莼(Ulya pertusa)凝集素的分离纯化及性质的研究

为抑制肿瘤细胞增殖和防治有关病害提供基础理论依据,将孔石莼(Ulva pertusa)经磷酸盐缓冲液抽提,20%～75%硫酸铵分级沉淀,牛甲状腺球蛋白-Sepharose 4B亲和层析,可以从绿藻孔石莼中纯化出孔石莼凝集素(UPL),在PAGE上显示单一蛋白染色带,在等电聚焦电泳上显示单一蛋白染色带,其pI为8.40.纯化后的UPL的最大紫外吸收峰在285 nm,用Sephadex G-200分子筛层析测得其分子量为11 047.该凝集素可以凝集人的A、B、AB、O型红细胞,且凝集活性相同,在对人(A、B、AB、O)兔、鲤、鲫的红细胞的凝集作用中,兔的凝集作用最强.该凝集素凝集兔红细胞的作用不被D-半乳糖、D-果糖、葡萄糖、蔗糖、甘露聚糖、γ球蛋白、卵清蛋白所抑制,仅被牛甲状腺球蛋白抑制,最小抑制浓度为6.20 g/L.该凝集素在pH4.0～10.14范围内均有活性,但在pH6.50～9.51范围内活性较高,该凝集活性在85℃加热1 h,活力仍未改变,说明具有很强的耐热性.     

从绿藻礁膜（Monostroma nitidum Wittr）分离半乳糖专一的凝集素

礁膜（Monostroma nitidum Wittr）经 25%～80%硫酸铵分级、DEAE-纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到纯化礁膜凝集素（Monostroma nitidum lectin,MNL）,在SDS-PAGE上显示单一蛋白染色带. 用Sephadex G-200层析测得其分子质量为66.6 kD, 用SDS-PAGE测得其分子质量为66.2 kD．该凝集素可以凝集人A、B、AB、O型红细胞,且凝集活性相同. 在对人（A、B、AB、O）、兔、鲤、鲫、鼠、羊、鸡、狗的红细胞凝集作用中,兔凝集作用最强．该凝集素在pH 4.00～10.53范围内均有活性,但在pH 5.20～9.40范围内活性最大．经100 ℃热处理30 min后,该凝集素对兔红细胞血凝活性保留25%,活性最大的温度范围为25～55 ℃．MNL被EDTA抑制,最小抑制浓度为3.13 mmol/L,但对 Ca^２＋和Mg^２＋不敏感．该凝集素凝集兔红细胞的作用不被D -果糖、D-甘露糖、D-葡萄糖、蔗糖、麦芽糖、γ-球蛋白、牛甲状腺球蛋白所抑制,但被D- 半乳糖和乳糖抑制,最小抑制浓度分别为5 mmol/L和2.5 mmol/L．     

红藻条斑紫菜凝集素的纯化及其色氨酸化学修饰对其活性的抑制作用

为了探索条斑紫菜凝集素(Porphyra yezoensis Ueda lectin, PYL)的作用机理,对其进行了分离和纯化．条斑紫菜经磷酸盐缓冲液浸泡、20%～75%硫酸铵分级、DEAE 纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到PYL纯品. Sephadex G-200分子筛层析测得其分子量为63.2 kD,在非还原SDS-PAGE上显示1条蛋白染色带,分子量为63.1 kD,还原SDS-PAGE显示1条蛋白染色带,亚基分子量为15.8 kD．PYL在对兔、大鼠、鸡、羊、狗血细胞的凝集作用中,对大鼠红细胞的凝集活性最高.PYL在pH 6.50～10.53范围内均有活性,在pH 8.40～8.91活性最高．经42 ℃热处理10 min后,仍然对大鼠红细胞血凝活性保留12.5%,其活性最大温度范围为4 ℃～20 ℃, 48 ℃加热10 min后,其活性完全丧失．EDTA对PYL的凝集活性有抑制作用,最小抑制浓度为156 mmol/L,而 Ca2+和Mg2+未发生凝集抑制现象．PYL凝集大鼠红细胞的作用不被D 果糖、葡萄糖、D-半乳糖、D-甘露糖、菊粉、γ球蛋白、牛甲状腺球蛋白等所抑制,但可被蔗糖和麦芽糖抑制,最小抑制浓度蔗糖为20 mmol/L,麦芽糖为40 mmol/L．用N 溴代丁二酰亚胺(NBS) 对PYL分子中的Trp残基进行化学修饰,有2.1个Trp残基被修饰,修饰后PYL活性丧失, 表明Trp残基是PYL凝集活性所必需的基团．     

岩豆(Millettia dielsiana Harms ex Diels)凝集素的分离纯化和性质研究

用猪甲状腺球蛋白-Sepharose 4B作亲和吸附剂,再经Sephadex G-100凝胶过滤,可以从岩豆种子中纯化出岩豆凝集素(MDL)。该凝集素可以凝集人类A、B、O型血细胞和兔红细胞,纯化的MDL凝集兔红细胞的能力可被D-松三糖、邻硝基-苯酚-D-半乳糖和N-乙酰半乳糖胺抑制,甘露糖也有弱的抑制作用。纯化的MDL在PAGE和SDS-PAGE上均显现单一蛋白质染色带,经Schiff’s试剂染色证明为糖蛋白;以酚-硫酸法测得其中性糖含量为6.0％;SDS-PAGE测得亚基分子量为32 000;Sephadex G-100分子筛柱测得其分子量为63 800;等电聚焦电泳显示其等电点为5.1;氨基酸组成分析表明其中Asp、Glu、Phe含量较高,但不含有Pro、Tyr。MDL也是一个强促有丝分裂原,对人外周血淋巴细胞转化率可达81.2％,细胞分裂比率达14.8％。     

梨形环棱螺凝集素的初步研究

通过Sepharose 4B-甲状腺球蛋白亲和层析,从梨形环棱螺Bellamya purificata体内分离到的一种凝集素,不连续PAGE显示其为单一的蛋白质谱带.它能凝集兔、猪、鸭等动物的红细胞,但不能凝集人的A、B、O及AB型血的红细胞和固定后的兔红细胞.其凝集活力可被1.0mol/L的乳糖、半乳糖和60g/L的甲状腺球蛋白抑制,但不能被碱性硼酸缓冲液抑制.对温度变化敏感,有较宽的最适pH范围.     

黄精凝集素Ⅱ的纯化及部分性质研究

囊丝黄精（ＰｏｌｙｇｏｎａｔｕｍｃｙｒｔｏｎｅｍａＨｕａ．）的根状茎,经浸取、用硫酸铵分级沉淀、猪甲状腺球蛋白－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析、ＣＭ－Ｓｅｐｈａｒｏｓｅ柱离子交换层析和ＳｅｐｈａｄｅｘＧ－１００凝胶过滤,可以分离纯化出黄精凝集素Ⅱ（ＰＣＬⅡ）．纯化的ＰＣＬⅡ在聚丙烯酰胺凝胶电泳中显示单一蛋白染色带;在快速高效液相色谱中亦为单一蛋白峰,经分子筛层析测得分子量为１５．９ｋＤ,最大紫外吸收值在２７８ｎｍ,ＰＣＬⅡ只凝集兔红细胞,当浓度为０．２５μｇ／ｍｌ时,即可发生凝集反应,此凝集兔红细胞的能力可被Ｄ－甘露糖和猪甲状腺球蛋白所抑制．氨基酸组成分析表明ＰＣＬⅡ分子中富含酸性氨基酸,Ｎ末端为丙氨酸．经测定ＰＣＬⅡ分子中含有３个色氨酸和２．４％的中性糖．原子发射光谱分析表明,该凝集素分子中含有Ｍｇ和Ｃａ两种金属元素．     

一株产凝集素的三叶半夏内生真菌的研究

研究三叶半夏内生真菌及其凝集素,旨在为半夏内生真菌及其凝集素的开发利用提供依据。对三叶半夏块茎内生真菌分离、纯化,液体发酵培养代谢产物,无水乙醇提取总蛋白,兔血红细胞检测其凝集活性,筛选出菌株gs1,其总蛋白对兔血红细胞凝集活性显著。使用甘露聚糖-Sepharose 4B亲和层析柱纯化菌株gs1总蛋白,得到凝集素。Brandford法定量检测分析表明,1000 ml gs1发酵培养液中含有9.58 mg 凝集素。SDS-PAGE 电泳分析显示该凝集素为单一条带,分子量约为12 kDa。凝集活性实验表明,该凝集素对兔、大鼠和小鼠的血红细胞具有凝集作用,对兔血红细胞效果最显著;而对人（A＼B＼O＼AB型）和鸡的血红细胞无凝集作用。糖结合活性实验表明,甘露糖对该凝集素的凝集活性具有抑制作用。通过初步分类鉴定,菌株gs1为半知菌亚门,丝孢纲,丛梗孢目,丛梗孢科,曲霉属。     

黑色菜豆(Phaseolus sp.)凝集素的纯化和性质

黑色菜豆(phaseolussp.)种子中含有对人A型血专一凝集的凝集素。用猪胃粘蛋白-Sepharose 4B作亲和吸附剂和Sephadex G-200凝胶过滤,可以纯化这种凝集素。纯化的凝集素在pH8.9,Tris-EDTANa_2-borate缓冲液的PAGE中,呈现单一蛋白带;酚-硫酸法测得总糖含量为3.22％。在SDS-PAGE中发现其分子由两种亚基所组成,亚基分子量分别为38,000和35,000。当凝集素浓度分别为0.98μg/ml和1.95μg/ml时能强烈地凝集人A型和AB型血细胞。在凝集素浓度高达500μg/ml时,B型血细胞能发生弱凝集反应,但对O型血和兔红细胞则完全不发生凝集反应。其凝集活性可被GalNAC、L-Fuc、猪甲状腺球蛋白和卵粘蛋白所抑制。该凝集素对人外周血中淋巴细胞的转化率达80％,细胞分裂比率高达37.1％;氨基组成分析表明,凝集素分子中Asp和Glu含量较高,而cys和Met含量很低。     

染病刺参凝集素的分离纯化与性质比较

目的：研究刺参免疫机制,探寻其发病机理。方法：刺参（Apost／chopusjapon／cus）经磷酸盐缓冲溶液抽提、20—75％硫酸铵分级沉淀、DEAE—SepharoseF．F．和SephadexG一100柱层析,得到刺参凝集素（A几）。结果：经SDS—PAGE显示单一条带,亚基相对其分子量为31000。分别提取感染Tenacibaculum属和Vibrionaks属两种刺参烂皮病病原菌的刺参凝集素,感染Temcibaculum属病菌刺参的凝集素产量较大,但凝集兔红细胞的作用均不被所测试的单糖和寡糖所抑制,而仅被牛甲状腺球蛋白所抑制。凝集兔红细胞的作用均不被二价金属离子Ca2＋、Mg2＋、Mn2＋、Zn2＋及EDTA所抑制。在pH4．0。10．14系列缓冲液中均有活性,其中在pH4．0—7．5时活性最大。该凝集活性在90℃加热30min后凝集活性仅损失50％。结论：推断菌株Tenac／bacu／um可诱导刺参体内产生大量的凝集素。     

棕尾别麻蝇(Sarcophaga peregrina)幼虫与蛹血淋巴凝集素的纯化与特性

用亲和层析法纯化了棕尾别麻蝇幼虫和蛹血淋巴凝集素。以兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗体、球球蛋白和甲状腺蛋白等三种亲和层析吸附剂纯化得到的幼虫凝集素是相同的,其分子量73kD左右。用甲状腺球蛋白为亲和配基纯化的蛹血淋巴凝集素由二种亚基组成,其分子量分别为30和32kD。幼虫和蛹血淋巴凝集素活性的抑制糖明显不同：乳糖、岩藻糖和N－乙酰半乳糖胺对幼虫血淋巴凝集素活性有抑制作用;而甘露糖胺、半乳糖胺和葡萄糖胺则对蛹血淋巴集素有一定抑制。而且,用兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗血清对蛹的凝集素活性无交叉反应,表明这两种凝集素是不相同的。虽然本文所纯化的麻蝇蛹血淋巴凝集素的分子量和Komano等报道的麻蝇蛹以及幼虫体壁 伤害诱导的凝集素SPL相同,但其糖的抑制特性有明显差异。     

Molecular characterization of a new lectin from the marine alga Ulva pertusa

A new lectin, named UPL1, was purified from a green alga Ulvapertusa by an affinitychromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectinwas about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinatingactivity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. Thelectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and hadhigher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined(P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned byrapid amplification ofcDNA ends (RACE) method (AY433960). Sequence analysis of upll indicated it was! 084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the matureUPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 aminoacids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not showamino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigmof a novel lectin family.     

Purification,characterization and induction of a C-type lectin in the freshwater planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

Lectins are important components of the immune defense system of invertebrates. Given their important functions, numerous investigations have been carried out on the characterization and function of lectins in invertebrates. However, lectin studies with the freshwater planarian, an evolutionarily important animal, are rare. In this paper, we demonstrate agglutination of glutaraldehyde treated erythrocytes by a lectin with preference for rabbit erythrocytes. The result of hemagglutinating activity inhibition assays with several carbohydrates showed the most potent inhibitor was maltose. A natural lectin from the crude homogenates of freshwater planarian Dugesia japonica was purified by single step affinity chromatography using amylose-coupled agarose. The purified protein appeared as one band with a molecular mass of 350 kDa in PAGE, and as one band, approximately 56 kDa, in SDS-PAGE. The purified lectin showed dependence on calcium. The activity of the purified lectin was inhibited at temperatures greater than 50°C and showed a pH optimum between 5–8. The purified lectin also has binding activity to the Gram-negative bacteria E. coli, and the Gram-positive bacteria B. subtilis. Furthermore, the purified lectin obtained from injured and bacteria-induced planarians showed increased agglutinating activity against rabbit erythrocytes. These results suggest that the purified lectin may play an important role in the innate immunity of the freshwater planarian.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca(2+)-dependent. The hemagglutinating activity of FmL was stable up to 55 degrees C and at pH 7.5-8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

泥蚶(Tegillarca granosa Linnaeus)血淋巴液凝集素的分离纯化及其性质研究

泥蚶是一种重要的海产经济贝类,其血淋巴液经硫酸铵二步分级沉淀后,再经Sephadex G- 100凝胶过滤和Sepharose 4B亲和层析纯化制得泥蚶血淋巴液凝集素。经测定,该凝集素分子量约为123Kda,为两个亚基的蛋白质,其相对分子量分别为15 KDa和16 KDa,分子中含5．02％的糖。在氨基酸组成中,天门冬氨酸(Asp)含量最高,其次是谷氨酸(Glu)和组氨酸(His),不含蛋氨酸(Met)。泥蚶血淋巴液凝集素对多种天然或经酶修饰的人或动物红细胞具有不同的凝集作用,其中对兔红细胞的凝集活性最大。半乳糖和乳糖对其凝集活性具有抑制作用。凝集活性依赖于Ca2 ,在pH7．0较稳定,热稳定性不高,在30℃-70℃时凝集效价由原来的25下降为21,当温度超过80℃以后,凝血活性完全丧失。     

Purification and characterization of a mannose-binding lectin from the rhizomes of Aspidistra elatior Blume with antiproliferative activity

A lectin with a novel N-terminal amino acid sequence was purified from the rhizomes of Aspidistra elatior Blume by ammonium sulphate precipitation, ion exchange chromatography on diethylaminoethyl-Sepharose and carboxymethyl-Sepharose and gel filtration chromatography on Sephacryl S-100. The A. elatior Blume lectin （AEL） is a heterotetramer with a molecular mass of 56 kDa and composed of two homodimers consisting of two different polypeptides of 13.5 kDa and 14.5 kDa held together by noncovalent interactions. Hapten inhibition assay indicated that hemagglutinating activity of AEL towards rabbit erythrocytes could be inhibited by D-mannose, mannan, thyroglobulin and ovomucoid. The lectin was stable up to 70 ℃ , and showed maximum activity in a narrow pH range of 7.0-8.0. Chemical modification and spectrum analysis indicated that tryptophan, arginine, cysteine and carboxyl group residues were essential for its hemagglutinating activity. However, they might not be present in the active center, except some carboxyl group residues. AEL also showed significant in vitro antiproliferative activity towards Bre-04 （66%）, Lu-04 （60%） and HepG2 （56%） of human cancer cell lines.     

Purification and some properties of hemagglutinating protein mutina from bushmaster Lachesis muta snake venom

Lachesis muta snake venom induced aggregation of bromelain sensitized human erythrocytes at a concentration of 1 mg/ml. The hemagglutinating protein was purified by DEAE-Sephadex A-50 column chromatography. Polyacrylamide gel electrophoresis revealed at least three bands, whereas SDS electrophoresis in the presence of 2-mercaptoethanol showed a single one. Isoelectric focusing revealed hemagglutinating activity in the range of pH 3-8. The maximum peak (mutina) at pH 5.5. This fraction was active in agglutinating human RBC of types A, B, O Rh (+) and B, O Rh (-). One mM EDTA and 1 mM Ca++ did not alter the agglutinating time significantly. Lactose and inositol inhibited the agglutination of A, B, O Rh (+) and B, O Rh (-) human RBC. The present study showed the non specificity of the hemagglutinating activity of mutina. It was also shown that mutina is a non-mitogenic protein.     

Pollen hemagglutinating activity is not related to lectin

We purified a hemagglutinating substance from pollen extracts to electrophoretic homogeneity. The hemagglutinating activity of this substance was inhibited by positively charged substances but not by the monosaccharides known as inhibitors of the lectin activity. The hemagglutinating substance appeared to be negatively charged in the pH conditions of the hemagglutinating assay, suggesting that hemagglutination was due to an electrostatic interaction with positively charged components of the erythrocytes. Preliminary chemical analysis suggests that the hemagglutinating substance is a proteoglycan.