红花菜豆凝集素的寡糖顺序分析

纯化的红花菜豆凝集素经肼解,Ｂｉｏ－ＧｅｌＰ－２柱分离,得到的寡糖部份用硼氢化钠还原,再红Ｂｉｏ－Ｇｅｌ Ｐ－４柱层析,获得两条寡糖链。通过甲醇解,乙酰化反应,最后制成三甲基硅醚衍生物。气相色谱分析表面两个寡糖的组成为;Ｍａｎ８Ｘｙ１ＧｌｃＮＡｃ２和Ｍａｎ６ＸｙｌＧｌｃＮａ２Ｆｕｃ。     

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．     

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     

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     

人参培养细胞中活性六糖的分离及结构鉴定

将用酸水解人参细胞方法得到的寡糖素,是一组聚合度由３－１２单糖组成的水溶性混合物,经活性碳柱,ｏｗｅｘ５０（Ｈ＾＋）柱,Ｂｉｏ－Ｇｅｌ Ｐ－２凝胶柱后由半制备型ＯＤＳ柱在ＨＰＬＣ分离到一个六糖,通过ＧＣ,ＧＣ－ＭＳ,ＦＡＢ－ＭＳ和＾１３４Ｃ－ＮＭＲ测定,推测出了这个     

大熊猫乳酸脱氢酶同工酶H4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层分析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出乳酸脱氢酶同工酶Ｈ４,纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＡＰＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分了量为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     

大瓶螺凝集素的分离纯化及部分性质研究

大瓶螺蛋白腺经磷酸盐缓冲液抽提、硫酸铵分级沉淀、ＳｅｐｈａｄｅｘＧ－１００和Ｓｅｐｈａｒｏｓｅ４Ｂ凝胶过滤,可获得在不连续ＰＡＧＥ（ｐＨ４．３和ｐＨ８．９）上显示单一蛋白质染色带的大瓶螺凝集素（ＡＧＬ）．该凝集素对人血红细胞无血型专一性,但对Ａ型血红细胞的凝集作用最强．ＡＧＬ的血凝活力可被乳糖或半乳糖所抑制．ＡＧＬ分子中的中性糖含量为０．２４ｍｇ／ｍｇ蛋白质．用ＳＤＳ－ＰＡＧＥ法测得其亚基分子量为１５０００,且只有一种亚基．ＡＧＬ中Ｃｙｓ和Ｐｈｅ的含量较高,并较耐热．     

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．     

大瓶螺凝集素的分离纯化及部分性质研究

大瓶螺蛋白腺经磷酸盐缓冲液抽提、硫酸铵分级沉淀、Ｓｅｐｈａｄｅｘ Ｇ－１００和Ｓｅｐｈａｒｏｓｅ ４Ｂ凝胶过滤,可获得在不连续ＰＡＧＥ（ｐＨ４．３和ｐＨ８．９）上显示单一蛋白质染色带的大瓶螺凝集素（ＡＧＬ）。该凝集素对人血红细胞无血型专一性,但对Ａ型血红细胞胞的凝集作用最强。ＡＧＬ的血凝活力可被乳糖或半乳糖所抑制。ＡＧＬ分子中的中性糖含量为０．２４ｍｇ／ｍｇ蛋白质。用ＳＤＳ－ＰＡＧＥ法测得其亚基分     

尿激酶型纤溶酶原激活剂单克隆抗体的生产及其在抗原纯化中的应用

从ＣＨＯ工程细胞培养上清初步纯化的ｕＰＡ免疫ＢＡＬＢ／ｃ小鼠,通过杂交瘤技术制备单克隆抗体细胞株,取其中一株３８－１－７株作高密度大量培养,细胞密度达１３．２×１０６／ｍＬ时,抗体滴度为１∶６１．４４×１０４。用自制的ｕＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱纯化抗体。抗体滴度提高２４３倍。纯化后的抗体与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ珠交联,制成ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析抗体柱,亲和力常数：１．２８×１０９（ｍｏｌ／Ｌ）－１,交联率：８３．５％。直接从培养上清纯化ｕＰＡ,纯度为９６．３％,回收率：８１．６％±１９,纯化倍数：５０倍左右,比活１．１１±０．２９×１０５。试验结果表明该法效果好,方法简单、操作方便、值得进一步研究和应用。     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.     

Structures of the asparagine-linked sugar chain of glucose transporter from human erythrocytes

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.     

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.     

Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus.

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.     

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis

The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.     

Structural studies of the sugar chains of human parotid alpha-amylase

Human parotid amylase can be separated into three families of isoenzymes (A', A, and B) by Sephadex G-75 column chromatography. Isoenzymes in family B were free from carbohydrate, while those in family A were all glycoproteins. The carbohydrate moieties of family A isoenzymes were released from their polypeptide portions by hydrazinolysis and labeled by reduction with NaB[3H]4. The yield of total radioactive oligosaccharides indicated that family A isoenzymes all contain single asparagine-linked sugar chains in one molecule. The radioactive oligosaccharides were fractionated into one acidic and two neutral oligosaccharide fractions by paper electrophoresis and paper chromatography. By sequential exoglycosidase digestion in combination with a methylation study, their structures were determined to be: Gal beta 1 leads to 4 (Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 and 3[Gal beta 1 leads to 4 GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 (NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

New evidence of the substrate specificity of endo-beta-N-acetylglucosaminidase D

The susceptibility of a variety of oligosaccharides to endo-beta-N-acetylglucosaminidase D was investigated. The oligosaccharides having the structures of Man alpha 1----6 (GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, derived from complex type triantennary sugar chains, released +/- Fuc alpha 1----6GlcNAcOT upon incubation with the enzyme at almost the same rate as Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. When the reaction products were reduced with NaB3H4 and analyzed by Bio-Gel P-4 column chromatography, a new radioactive peak was detected in both cases. This new radioactive oligosaccharide was confirmed to be Man alpha 1----6(GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAcOT in the former case and Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAcOT in the latter. These results indicated that endo-beta-N-acetylglucosaminidase D does not require the presence of a free hydroxyl group at the C-4 position of the alpha-mannosyl residue of the trisaccharide glycon: Man alpha 1----3Man beta 1----4GlcNAc beta 1----.