Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora

Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.     

Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae.

The culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galbeta1 leads to 3GalNAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galbeta1 leads to 3GalNAc from glycopeptides and glycoproteins with Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr moieties. The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20 mM (on the basis of the amount of Galbeta1 leads to 3GalNAc residues) was obtained. So far, the enzyme appears to have a strict specificity for Galbeta1 leads to 3GalNAcalpha1 leads to Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.     

Purification of an exo-beta-D-glucanase from cell-free extracts of Candida utilis.

beta-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific beta-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-beta-linkages by an exo-splitting mechanism. Glucono-delta-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Blood group A activities of glycoprotein and glycolipid from human erythrocyte membranes.

It is known that ABO blood group substances in human erythrocyte membranes are sphingoglycolipids, but recently several authors have reported that the glycoproteins of the erythrocyte membranes also have ABO blood group activities in addition to MN blood group activities and virus hemagglutination inhibitor activity. We solubilized blood group A erythrocyte membranes with lithium diiodosalicylate and separated the glycoprotein fraction by phenol extraction and ethanol precipitation. This fraction was apparently not contaminated with glycolipid, but it showed weak blood group A activity. The activity of the glycoprotein of the erythrocyte membranes was one-sixth of that of the lgycolipid fraction from the same amount of membranes. The glycoprotein components were purified by Sephadex G-200 gel filtration in SDS. The main component isolated, PAS 1, still showed blood A activity.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes.

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed 'epithelial-membrane glycoprotein' (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.     

Isolation of Glycoproteins from Verticillium dahliae and Their Phytotoxicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes cotton wilt-disease. Glycoproteins secreted by V. dahliae have been found to play an important role in wilting syndrome. In this study the glycoproteins were purified consecutively by ConA-Sepharose 4B affinity chromatography, Sephadex G-150 gel filtration, two-dimensional gel electrophoresis and SDS gradient gel electrophoresis. The N-terminal residual sequence of a 26 kD glycoprotein was analyzed. Plant-wilting tests were carried out by injection of glycoproteins, and those treated by heat, ConA and zeatin, into cotton leaves, respectively. Results showed that heat and ConA treatment abolished the wilt-causing activity of the glycoproteins, and zeatin alleviated the wilt syndrome of cotton. Furthermore, the glycoproteins were found to be effective elicitors in inducing the biosynthesis of sesquiterpene aldehyde phytoalexins in suspension cell cultures of Gossypium barbadense L., and heat-treatment lowered, but not abolished the elicitor activity. However, application of native glycoproteins at the concentration higher than 5 mg/L resulted in cell death.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [³H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.     

Purification and characterization of the glycoprotein allergen from Prosopis juliflora pollen

Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen.     

Acidic protease from human seminal plasma. Purification and some properties of active enzyme and of proenzyme.

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Purification of a young age-related glycoprotein (Ugl-Y) from normal human urine

Ugl-Y is a glycoprotein that is detected in normal urine samples from young men and women aged 0 to 17 years. It was purified by ammonium sulfate precipitation and various column chromatographies including affinity chromatography using anti-adult urine antibody coupled to Sepharose 4B. The homogeneity of the glycoprotein was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, column chromatography on Sephadex G-75, and the precipitation reaction with anti-Ugl-Y antibody. It was shown to have a molecular weight of 29,000 by gel filtration, and to contain 5.2% neutral sugars (mannose and galactose) and 4% hexosamine (glucosamine). Amino acid analysis of the glycoprotein indicated high contents of acidic and hydroxylic amino acids. Its origin is unknown.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Isolation and various properties of thiamine-binding protein from synaptosomes in the rat brain

A thiamine-binding protein (ThBP) with a specific activity of 8.21 nmoles/mg protein was isolated from rat brain synaptosomes by affinity chromatography and gel filtration on Sephadex G-200. The protein was purified 746-fold with a 40.5% yield. ThBP was homogeneous during sodium dodecyl sulfate gel electrophoresis; its molecular mass was determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate gel electrophoresis and was equal to 107 and 103 kD, respectively. The pH optimum for the binding is 8.35. When the ability of ThBP to bind thiamine phosphates was tested, the latter decreased in the following order: thiamine monophosphate greater than thiamine triphosphate greater than greater than thiamine diphosphate.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.     

Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.     

Composition and distribution of carbohydrate chains in glycoproteins of human erythrocyte membrane

The M-, N-, and MN-glycoproteins obtained from human erythrocytes by phenol-water extraction were purified by gel filtration and digested with Pronase and trypsin. The products of degradation were fractionated by gel filtration on Sephadex G-25 and DEAE-Sephadex A-50 and the fractions were examined by poly(acrylamide)-gel electrophoresis in the presence of dodecyl sodium sulfate, analyzed for carbohydrate and amino acid contents, and tested for M and N blood-group activity. From the results, it is suggested that the glycoprotein chains are composed of a hydrophobic moiety devoid of carbohydrate chains and a hydrophilic moiety containing carbohydrate chains of different compositions, irregularly distributed along the protein chains and linked to L-asparagine, L-serine, or L-threonine residues. The M and N activity typical for the undegraded glycoproteins, and the “basic” or “precursor-type” N activity, were found in different glycopeptide fractions.