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α１－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     

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。     

鸡Zong凝集素的分离纯化与性质研究

鸡Ｚｏｎｇ菌丝体浸取液依次经硫酸铵分级沉淀,ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－１００分子筛层析３个主要步骤纯化得到一种凝集素（ＴＡＬ）。纯化的ＴＡＬ在聚丙烯酰胺凝胶电泳上显示一条蛋白质着色带。ＴＡＬ的分子量为８９．４ｋＤ,亚基分子量为３８ｋＤ和５１ｋＤ,提示ＴＡＬ分子由两个不同亚基组成。ＴＡＬ具有供血动物种属专一性,使Ｗｉｓｔａｒ大鼠红细胞凝集所需ＴＡＬ最     

笋瓜籽毒蛋白Maximin—一种新的蛋白质生物合成抑制剂的分离纯化…

笋瓜（Ｃｕｃｕｒｂｉｔａ ｍａｘｉｍａ）种籽经去壳捣碎,乙醚脱脂,硫酸铵分级沉淀,阳离子交换层极及凝胶过滤等步骤,纯化到一种有蛋白质生物合成抑制活性的碱性蛋白,命名为Ｍａｘｉｍｉｎ,ＳＤＳ－ＰＡＧＥ显示单一条带,其分子量约为２８ｋＤ。该蛋白对兔网织红细胞裂解液的蛋白生物合成有较强的抑制,ＩＣ５０为４．０×１０＾－１０ｍｏｌ／Ｌ,与天花粉蛋白（ＴＣＳ）毒性相似。从这些特性可见,该蛋白似为单链核糖体失     

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     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

以痘苗病毒天坛株为载体的表达单纯疱疹病毒Ⅰ型糖蛋白D的重组活疫苗的建立

从我国分离到的一株单纯疱疹病毒Ⅰ型（ＨＳＶ－１－１６８株）病毒基因组中,分离出含有糖蛋白Ｄ（ｇＤ）基因的１．２ｋｂ片段,插入带有痘苗病毒天坛株ＴＫ区的质粒ｐＪＳＢ１１７５Ｐ７．５ｋ启动子下游,转染无白血病鸡胚原代细胞,获得带有ＨＳＶ－１－１６８ｇＤ基因的重组痘苗病毒。此株重组病毒在感染细胞膜上表达ＨＳＶ－１－１６８ｇＤ糖蛋白抗原,能与特异性单克隆抗体反应。在感染细胞中表达的膜抗原经ＳＤＳ－ＰＡＧＥ分析,表达分子量为５４ｋＤ糖蛋白。用Ｓｏｕｔｈｅｒｎ杂交分析了重组病毒ＤＮＡ中特异的ｇＤ基因,对作为活疫苗的重组痘苗病毒株进行了一些微生物学活性、免疫原性和毒力等方面的研究。     

芦荟凝集素的分离、纯化和部分性质的研究

新鲜芦荟叶（Ａｌｏｅ ｖｅｒａ Ｌ．ｖａｒ．ｃｈｉｎｅｎｓｉｓ（Ｈａｗ．）Ｂｅｒｇｅｒ）于室温用低浓度ＮａＣｌ溶液提取。离心和透析后,经Ｎ－乙酰氨基葡萄糖０Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,分离纯化出芦荟凝集素（ＡＣＬ）。用ＳｅｐｈａｄｅｘＧ－１００测表观分子量为３５ＫＤ,ＳＤＳ－ＰＡＧＥ出现两条色带;染色 ;宽带和较浅的罕带。亚基分子量分别为１５ＫＤ和２０ＫＤ。能专一性凝集兔血细胞和人血红细胞     

高分子量和低分子量尿激酶的分离纯化及动力学性质研究

人尿激酶粗品经苯甲脒亲和柱纯化和Ｐｒｏｔｅｉｎ－ＰａｋＳＰ柱分离后,得到两种分子量的尿激酶（ＵＫ）,即高分子量尿激酶（ＨＵＫ）和低分子量尿激酶（ＬＵＫ）,采用民斯亮蓝法测定蛋白质浓度,纤维蛋白板法测定活力,测得ＨＵＫ比活为２．９×１０＾５ＩＵ／ｍｇ蛋白,ＬＵＫ为３．５１０＾５ＩＵ／ｍｇ蛋白,活力回收为７０％以上,经ＳＤＳ－ＰＡＧＥ鉴定,ＨＵＫ和ＬＵＫ均呈单一条带,分子量分别为５４ｋＤ和３３ｋＤ,Ｈ     

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．     

Identification and characteristics of concanavalin A-binding neurospecific glycoproteins in human brain and brain tumors

Soluble and membrane-bound neurospecific Con A-binding glycoproteins from human brain and tumours were identified and characterized, using a procedure which included stepwise extraction with low and high ionic strength buffers, buffered. Triton X-100 and sodium deoxycholate followed by ConA-Sepharose column chromatography, SDS-PAAG electrophoresis and immunoblotting. Adsorbed antisera against different types of neurospecific glycoproteins were used. The bulk of neurospecific glycoproteins (11 and 13) were revealed in protein fractions extracted with low ionic strength buffers and Triton X-100. In astrocytomas and glyoblastomas, some neurospecific glycoproteins were absent. Some glycoproteins were found in tumours, but were absent in brain tissue. Soluble, 77 kD glycoprotein, 11 and 16 kD glycoproteins solubilized with high ionic strength buffers and intrinsic membrane-bound 51, 57, 61, 74 and 77 kD glycoproteins can be viewed as stable neurospecific markers in malignant brain tumours.     

高及低转移潜能小鼠肝癌细胞株糖蛋白凝集素结合特征的研究

为探讨肿瘤转移与细胞表面的糖结构的关系,对小鼠肝癌细胞的高、低淋巴道转移株Ｈｃａ－Ｆ和Ｈｃａ－Ｐ进行了蛋白质电泳及经蛋白质印迹术后的５种凝集素（ＣｏｎＡ、ＷＧＡ、ＵＥＡ、ＳＢＡ、ＰＮＡ）结合糖蛋白谱的对比分析．结果表明：高、低转移两株细胞的ＳＤＳ－ＰＡＧＥ谱基本相同;ＣｏｎＡ特异结合糖蛋白共有５种（～７２,８０～９０,～１０４,～１５０,～２００ｋＤ）;其中较明显的差异为～７２ｋＤＣｏｎＡ特异结合糖蛋白,它在Ｈｃａ－Ｐ细胞的表达明显高于Ｈｃａ－Ｆ细胞．ＷＧＡ特异结合糖蛋白１种（～１５０ｋＤ）,在Ｈｃａ－Ｐ细胞的表达略高于Ｈｃａ－Ｆ细胞．此外,实验发现两种性质未明的蛋白质（～７９,～１３０ｋＤ）,后者在Ｈｃａ－Ｐ细胞的含量明显高于Ｈｃａ－Ｐ细胞．结果提示Ｈｃａ－Ｆ和Ｈｃａ－Ｐ细胞不同的转移表型可能与其糖蛋白的表达有一定的关联．     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

Differential expression of sialic acid and N-acetylgalactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential.

In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions of the cells. Fluorescence and electron microscopic analyses revealed differences in the expression profile of carbohydrates recognized by the lectins used on the cell surface of IEC-6, Caco-2, and HCT-116 cells. Lectin blotting identified a range of eight HPA-binding glycoprotein bands with molecular weights of 16-66 kD in Caco-2 cells, six glycoproteins of 16-36 kD, and three protein bands of 35, 24, and 21 kD in IEC-6 cells. A minor band of 66 kD and a major one of 50 kD for WGA-binding glycoproteins were observed in IEC-6 cells and seven glycoproteins of 18-97 kD in Caco-2 and HCT-116 cells but with a visible higher expression of these glycoproteins in the latter. Furthermore, significant quantitative difference in levels of expression of WGA- but not of HPA-binding glycoconjugates was noted, as analyzed by high-resolution scanning electron microscopy using backscattered electron images of cells incubated with gold-labeled lectins.     

Association of specific cell-surface glycoproteins with a triton X-100-resistant complex of plasma membrane proteins isolated from T-lymphoma cells (P1798)

A non-ionic detergent-resistant complex of membrane-associated proteins and cell-surface glycoproteins has been isolated by gel filtration and isopyknic centrifugation of purified plasma membranes from the murine T-lymphoma P 1798. This complex elutes as a high molecular weight peak (greater than 15 X 10(6) D) and contains two specific sets of (1) cell-surface glycoproteins; (2) membrane-associated proteins. The cell-surface glycoproteins consist of two vectorially labelled major components present in a fixed molar ratio: The Thy-1 glycoprotein and a non-H-2 glycoprotein of 55 kD. Minor but significant amounts of the class I histocompatibility antigen Qa-2 are also contained in the detergent-resistant complex. The membrane-associated proteins are not vectorially labelled, and form a complex group of proteins in the 30-70 kD range. Since actin is not detectable among these polypeptides, they probably constitute a plasma membrane-associated structure that is distinct from actin-containing, submembranous cytoskeletal elements.     

Plasma membrane glycoproteins of tumour cells in enzootic bovine leukosis compared with normal bovine lymphoid cells

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.     

Isolation and characterization of blood group N antigen precursor glycoproteins with Thomsen-Friedenreich (T) activity, N antigen precursor glycoproteins and T-active glycoproteins from cyst fluids of malignant ovarian clear cell carcinoma

1. Two perchloric acid-soluble glycoprotein fractions from cyst fluids of 2 patients with ovarian clear cell carcinoma in malignant were subjected to a systematic affinity chromatography using Vicia unijuga lectin-Cellulofine column and Arachis hypogaea lectin-Cellulofine column and separated into four glycoproteins, blood group N antigen precursor glycoprotein with Thomsen-Friedenreich (T) activity, N antigen precursor glycoprotein, T-active glycoprotein and serologically inactive glycoprotein, respectively. 2. These serologically active glycoproteins isolated in yields of 0.3-3.3% of PASFs, were proved to be mucin-type glycoproteins with Mw of 509,000-2,772,000 Da and contained 33.9-81.7% carbohydrates.     

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

Polypeptides of mumps virus.

Mumps virus was propagated in the extra-embryonic fluids of embryonated chicken eggs and was labeled by cionjection of radioactively labeled amino acids. The virus was purified by density gradient centrifugation, and its polypeptides were analyzed by polyarylamide gel electrophoresis. The virus was found to be composed of six polypeptides, ranging in size from 40,000 to 64,000 daltons. Viral proteins 1 and 3 were the glycoproteins of the virons. When the virus particle was treated with noniontic detergents, a small fraction of these glycoproteins could be released into the supernatant. After treatment with nonionic detergents in high salt and alkaline conditions, more of the surface glycoproteins were removed. This treatment also released the smallest viral polypeptide from the virion. The glycoproteins were separated using an affinity chromatographic column of agarose-fetuin. The heavier glycoprotein, viral protein 1, was found to contain both the neuraminidase and hemagglutinating activity. The two glycoproteins were tested for their ability to react in complement-fixing tests with mumps antisera. Only the heavier glycoprotein reacted with antisera possessing both anti-S and anti-V activity. Neither glycoprotein reacted with antisera specific for the S antigen. Thus, it was concluded that this glycoprotein corresponds to the classical V antigen of mumps virus.