天花粉中三个同工凝集素的分离纯化及其生物学性质研究

运用阴离子交换层析从亲和纯的天花粉凝集素中分离得到三个同工形式的凝集素,它们都具有结合半乳糖的能力,都能与天花粉凝集素的的因清形成免疫沉淀线,并且其它各方面的性质如疏水结合能力也相仿,对它们生物学性质的检测,发现它们都能杀伤黑色素瘤细胞,但缺乏抑制无细胞蛋白质生物合成的能力,另外,对植物中同工形式凝集素的存在进行了讨论。     

天花粉凝集素糖结合专一性Ⅱ.

本文报道一些糖类、糖苷、糖蛋白和几种外源凝集素对标记天花粉凝集素和酸处理交联琼脂糖或胎盘细胞膜结合的影响。在低浓度时,所用的糖类中,乳糖是最强的抑制剂,蜜二糖和棉籽糖的抑制能力和乳糖相仿,而纤维二糖、蔗糖、麦芽糖,则无明显影响。三个所用的糖蛋白,它们的抑制活性以下列顺序递减:猪甲状腺球蛋白,人血清转铁蛋白,鸡卵白蛋白。未标记的天花粉凝集素和蓖麻凝集素,两者都专一地和半乳糖结合,它们都能竞争标记天花粉凝集素,而伴刀豆球蛋白A和半夏凝集素则不能竞争。由此,我们推测天花粉凝集素主要是和半乳糖结合,但与乳糖的结合能力最强,故推测其结合部位能容纳半乳糖和另一个单糖。     

天花粉凝集素糖结合专一性Ⅱ.

本文报道一些糖类、糖苷、糖蛋白和几种外源凝集素对标记天花粉凝集素和酸处理交联琼脂糖或胎盘细胞膜结合的影响。在低浓度时,所用的糖类中,乳糖是最强的抑制剂,蜜二糖和棉籽糖的抑制能力和乳糖相仿,而纤维二糖、蔗糖、麦芽糖,则无明显影响。三个所用的糖蛋白,它们的抑制活性以下列顺序递减:猪甲状腺球蛋白,人血清转铁蛋白,鸡卵白蛋白。未标记的天花粉凝集素和蓖麻凝集素,两者都专一地和半乳糖结合,它们都能竞争标记天花粉凝集素,而伴刀豆球蛋白A和半夏凝集素则不能竞争。由此,我们推测天花粉凝集素主要是和半乳糖结合,但与乳糖的结合能力最强,故推测其结合部位能容纳半乳糖和另一个单糖。     

天花粉同工凝集素—1双链的拆分和鉴定

天花粉同工凝集素－１经巯基乙醇还原,碘代乙酰胺保护,其链间二硫键被打开,但仍非共价结合在一起。我们利用尿素变性的Ｑ－Ｓｅｐｈａｒｏｓｅ离子交换层析分离了此凝集素的两条链。氨基酸组成测定与其他３种肽链作一比较,它们都含有较多的酸性和羟基氨基酸。蛋白质印迹显示ＴＫＬ的抗血清不仅能与ＴＫＬ－１的两条链分别分应,也能与天花粉毒蛋白及蓖麻毒蛋白的Ａ链起作用。溴化氰裂解的ＳＤＳ－ＰＡＧＥ肽谱表明天花粉凝集素的     

天花粉蛋白对体外培养不同种类细胞的作用

本文进行了天花粉蛋白对离体培养的葡萄胎细胞、早孕蜕膜细胞、人胎儿肾皮质部细胞、HeLa细胞和鼻咽癌上皮细胞的损伤作用的比较研究。为了探索天花粉蛋白选择损伤细胞是否存在专一结合细胞的可能性,又以不敏感的蜕膜细胞为材料,应用辣根过氧化物酶标记技术进行了天花粉蛋白与细胞结合和进入细胞内定位的实验。此外,并以肝癌细胞为材料研究了对细胞增殖率的影响。结果表明葡萄胎合体滋胚层细胞和胎盘合体滋胚层细胞一样都是最敏感的细胞,经1微克天花粉蛋白处理即出现形态上损伤,至50微克则大多数细胞呈现不可逆的退化损伤,而其它类型的细胞则表现出有相当大的抗性。过氧化物酶标记追踪天花粉蛋白在细胞内分布的结果表明天花粉蛋白与蜕膜细胞有一定的亲和力,提示天花粉蛋白似乎不存在严格的细胞结合专一性;而天花粉蛋白在细胞内的分布一般仅限于细胞质,这一现象似乎提示细胞质区可能就是天花粉蛋白直接作用的靶的范围。细胞增殖率的实验结果表明50微克以上的药物剂量对肝癌细胞增殖率有一定的抑制作用,剂量增加抑制作用也增加。但是,经过一定的修复期以后,细胞增殖率又可得到完全恢复,反映了对细胞生长抑制的可逆作用。上述实验结果进一步加强了天花粉蛋白对滋胚层细胞具有一定细胞损伤专一性的结论。本文并对细胞损伤专一性问题进行了讨论。     

天花粉蛋白对滋养层细胞专一损伤机制的研究

在体外培养条件下,天花粉蛋白胶体金以受体介导的内吞方式进入滋养层细胞和绒癌细胞,最终进入胞质溶胶附着在核糖体上。经相同处理的肝癌细胞,绿猴肾细胞和正常鼠胚肝细胞,金颗粒既不与这些细胞表面结合,也没有被专一内吞的现象。分别用BSA,转铁蛋白活化的胶体金处理滋养层细胞和绒癌细胞的比较观察,也证明滋养层细胞和绒癌细胞对天花粉蛋白的专一亲和性。更有趣的是:天花粉蛋白肝癌单抗胶体金结合物进入绒癌细胞的方式与天花粉蛋白的结果相同;先以旰癌单抗处理也不影响天花粉蛋白肝癌单抗结合物进入绒癌细胞。然而,对于旰癌细胞,天花粉蛋白肝癌单抗胶体金颗粒则专一地结合在细胞的微绒毛表面,这种结合因先以肝癌单抗处理而明显地被竞争抑制。这些实验结果相互印证地提供天花粉蛋白对滋养层细胞和绒癌细胞高度专一的亲和性,并证明天花粉蛋白对靶细胞的原初损伤部位是核糖体。进一步探讨天花粉蛋白在靶细胞表面结合位点的化学性质以及这种蛋白在靶细胞内的转运机制将是重要的问题。     

天花粉同工凝集素－1双链的拆分和鉴定

天花粉同工凝集素－１经巯基乙醇还原,碘代乙酰胺保护,其链间二硫键被打开,但仍非共价结合在一起。我们利用尿素变性的Ｑ－Ｓｅｐｈａｒｏｓｅ离子交换层忻分离了此凝集素的两条链。氨基酸组成测定与其他３种肽链作一比较,它们都含有较多的酸性和羟基氨基酸。蛋白质印迹显示ＴＫＬ的抗血清不仅能与ＴＫＬ－１的两条链分别反应,也能与天花粉毒蛋白及蓖麻毒蛋白的Ａ链起作用。溴化氰裂解的ＳＤＳ－ＰＡＧＥＲ肽谱表明天花粉凝集素的两条链与天花粉毒蛋白含有类似的裂解片段,在分子量１６ｋｄ左右有相同的电泳条带。ＴＫＬ－１两亚基的Ｎ末端序列已经测定,同源性比较发现其３３ｋｄ亚基的Ｎ末端序列与天花粉毒蛋白、蓖麻毒蛋白的一些肽段类似。迄今已有的证据表明ＴＫＬ与ＴＣＳ等是一些非常相关的蛋白质。     

小鼠抗天花粉蛋白IgE单克隆抗体的制备、分离和鉴定(英文)

天花粉蛋白是中药天花粉的有效引产成份,在应用中它偶尔也引起过敏反应。我们用杂交瘤技术建立了一株分泌抗天花粉蛋白特异性IgE单克隆抗体的杂交瘤细胞系。在实验中,二次免疫的C57 BL/6 J小鼠的肠系膜淋巴结细胞和脾细胞分别被用来同NSI骨髓瘤细胞进行融合。虽然在融合前动物血清IgE抗体效价仅为40～160 PCA滴度,但用肠系膜淋巴结细胞进行的4次融合都产生了IgE杂交瘤。阳性率为1.0-6.7%。对比之下,用脾细胞进行的另外两次融合却没有观察到IgE杂交瘤产生,统计结果说明差异显著(P<0.05)。该IgE单克隆抗体可以在体内和体外诱发大鼠肥大细胞脱颗粒。56℃热处理2小时能使该抗体诱导PCA反应的能力完全丧失,然而却不影响其结合抗原的能力。对该单克隆抗体的特异性的鉴定表明,它可以特异性地识别精制天花粉蛋白和结晶天花粉蛋白上的抗原决定簇。     

坛紫菜凝集素的糖结合专一性和细胞凝集作用

坛紫菜的磷酸盐缓冲液浸取液,经硫酸铵沉淀和DEAE－Sepharose,SephadexG-100二步层析纯化,获得纯化的坛紫菜凝集素（PHL）。该凝集素能与3种单糖（阿拉伯糖,半乳糖,木糖）及麦芽糖专一性结合,其中与麦芽糖结合最强。细胞凝集实验结果显示,PHL能凝集兔,绵羊及鸡红细胞而不能凝集鸭,鸽子及人血红细胞,PHL还能凝集海洋微藻－绿色巴夫藻和淡水微藻－蛋白核小球藻,它们的凝集活性与藻细胞密度有关。不同状态的细菌和酵母细胞对PHL反应不同,表明随着细胞状态的改变,细胞表面的凝集素受体也随之发生变化。     

中性红染色后细胞对凝集素的反应性

中性红染色后的小鼠胸腺细胞。肝、脾和肾细胞,人扁桃体细胞,以及体外培养的不同肿瘤细胞(HL60,K562,Molt-4,MLA和L1210)都能够被凝集素凝集。除小鼠肝细胞和肾细胞外,它们的凝集特征都与红细胞的凝集特征相同。不同来源的细胞,其对凝集素的反应性不同,其中,以肿瘤细胞对凝集素的反应最敏感。结果说明,中性红染色法可以作为用凝集素初步检测一些无色细胞表面凝集素受体变化的一种简便方法。     

天花粉凝集素的荧光光谱研究

天花粉凝集素在天然状态下荧光发射峰位于３３２ｎｍ处,以丙烯酰胺,ＫＩ及ＣｓＣ１等淬灭剂研究ＴＫＬ分子中Ｔｒｐ残基的微环境,发现只有丙烯酰胺能淬灭ＴＫＬ分子Ｔｒｐ的荧光,同此推断大部分的Ｔｒｐ残基位于ＴＫＬ分子内部,其荧光不易为Ｉ＾－或Ｃｓ＾＋接近而淬灭。疏水探针ＴＮＳ能够检测到ＴＫＬ中疏水微区的存在,并且这一疏水微区亦不同于ＴＫＬ的半乳糖结合位点,ＴＫＬ中不存在金属离子的结合部位。     

Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.     

天花粉凝集素的固定化及其应用

制备了天花粉凝集素ＴＫＬ－Ｓｅｐｈａｒｏｓｅ４Ｂ的亲和柱,用来分离其它来源物质中与ＴＫＬ相互作用的糖复合物,发现从兔红细胞的血影细胞的抽提物中可以得到分子量为６２ｋＤ,４０ｋＤ等糖蛋白,这可能就是ＴＫＬ凝集兔红细胞的分子基础。从人及骆驼、绵羊血浆中也能得到与ＴＫＬ结合的、分子量不等的糖蛋白,进一步鉴定人血浆ＴＫＬ结合物中含有α＿１－抗胰蛋白酶等,为从血浆中分离这些糖蛋白提供了较为便利的方法。在寻找天花粉植物体内此凝集素的内源受体时,探讨了它可能的生理功能。     

Kluyveromyces bulgaricus yeast lectins. Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation

Kluyveromyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates. Its flocculation can be reversed by the addition of galactose. In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution. The N-acetylglucosamine-specific lectin, at pH 7.4, agglutinates untreated sheep red blood cells, but agglutinates neither untreated rabbit red blood cells nor glutaraldehyde-fixed sheep or rabbit red blood cells. Conversely, at pH 4.5, this lectin agglutinates glutaraldehyde-fixed sheep red blood cells. The galactose-specific lectin, at pH 7.4, agglutinates both untreated and glutaraldehyde-fixed rabbit red blood cells but does not agglutinate untreated or glutaraldehyde-fixed sheep red blood cells. At pH 4.5, this lectin agglutinates both glutaraldehyde-fixed sheep and rabbit red blood cells and induces flocculation of deflocculated K. bulgaricus cells. In all cases, the agglutination and the flocculation induced by one of these two lectins were inhibited by free or conjugated N-acetyl-D-glucosamine or by free or conjugated D-galactose, respectively. No glycosylhydrolase activity could be detected in the purified lectins.     

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.     

Isoforms of an endogenous lectin in rabbit bone marrow

A lectin which may mediate inter-erythroblast associations during red blood cell development in rabbit bone marrow has previously been purified and characterised. We have now detected different forms of this lectin in purified preparations and crude tissue extracts, by isoelectric focusing in agarose gels followed by rocket immunoelectrophoresis and by indirect antibody staining of focused proteins blotted onto nitrocellulose paper. These minor antigens are probably isoforms of the bone marrow lectin previously characterised.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Isolation and characterisation of a serum lectin from blue gourami, Trichogaster trichopterus (Pallus)

A novel lectin, designated BGL, has been purified from the serum of blue gourami, Trichogaster trichopterus, with the use of (NH4)2SO4 fractionation, affinity chromatography and gel filtration chromatography. Electrophoretic analyses and mass spectrometric study of purified BGL showed that the lectin is composed of two isoforms with native molecular masses estimated to be 65 and 66 kDa, and two subunits of 32 and 34 kDa on SDS-PAGE under non-reducing conditions. Upon reduction with 20 mM dithiothreitol (DTT), BGL showed two close bands of 27 and 29 kDa. After isoelectric focusing, the lectin focused as close double bands at pH 5.6. The N-termini of both isoforms share the same sequence (HGEENRXGPR) and show no significant homology with any known proteins. The BGL agglutinating activity is specifically inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine, and to a lesser degree by D-(+)-mannose, but not by D-(+)-galactose, D-(+)-glucose, maltose or N-acetyl-D-mannosamine. Haemagglutination assay showed that BGL is more specific for rabbit than mouse, chicken, rat or guinea pig erythrocytes, and haemagglutination was Ca2+-dependent. In addition, BGL could agglutinate a range of micro-organisms and yeast cells, with the exception of some fish pathogens, such as Aeromonas hydrophila (strains: PPD 134/91 and PPD 11/90) and Vibrio harveyi (strain: W618). Localisation of BGL by fluorescein isothiocyanate (FITC)-labelled antibodies revealed that the lectin is associated with the cell surface of fish leukocytes.     

Cumulus oophorus-associated glycodelin-C displaces sperm-bound glycodelin-A and -F and stimulates spermatozoa-zona pellucida binding

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.