Showing papers on "Affinity chromatography published in 1975"
TL;DR: A highly flexible method based on affinities which can be used in a more selective fashion by modern chromatographic techniques is described here.
Abstract: CONVENTIONAL nonspecific precipitation methods sometimes depend on affinities which can be used in a more selective fashion by modern chromatographic techniques. The affinity of proteins for heavy metal ions, for example, may provide a basis for their purification and analysis. A highly flexible method based on such affinities is described here.
2,063 citations
TL;DR: The lectin agglutinated erthrocytes of human ABO blood types equally well, but only after they have been treated with neuraminidase, suggesting that the four subunits are identical.
919 citations
TL;DR: It is found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column.
Abstract: Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.
403 citations
TL;DR: In this article, a procedure was described to utilize blue dextran-separharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins.
Abstract: A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins. The procedure can be used to identify proteins, either purified or in crude cellular extracts, that possess the dinucleotide fold and to significantly improve the purification procedures for those proteins that possess the fold.
360 citations
TL;DR: Electrolectin seems at least partially membrane-bound but is also found in soluble fractions of homoge-nates from which it can be purfied by affinity chromatography on cross-linked and desulfated agarose (ECD-Sepharose) as a protein of molecular weight 33,000.
Abstract: Extracts of electric organ tissue of Electrophorus electricus contain a saccharide-binding protein, named electrolectin, which agglutinates trypsin-treated rabbit erythrocytes and is specifically inhibited by disaccharides containing nonreducing terminal beta-D-galactosyl residues. Electrolectin seems at least partially membrane-bound but is also found in soluble fractions of homoge-nates from which it can be purfied by affinity chromatography on cross-linked and desulfated agarose (ECD-Sepharose) as a protein of molecular weight 33,000. About 400 mg of electrolectin are present per kg of tissue. It has an affinity for lactose of 1.0 mM-1 and 5.5mM-1 as estimated, respectively, by hapten inhibition and fluorescence spectroscopy. Studies on the distribution of beta-D-galactoside-binding activity in animal tissues reveal particularly high levels in sheletal muscle tissue and in cultures of embryonic skeletal muscle and neuroblastoma cells.
256 citations
TL;DR: The characteristics of peak 7 and peak 9 proteinosphorylation are similar to those of the platelet release reaction, suggesting that the phosphorylation of these proteins may play a role in the platelets release reaction.
Abstract: Intact human platelets loaded with 32PO4 contain multiple phosphorylated proteins. Thrombin treatment of intact 32PO4-loaded platelets results in a 2-6-fold increase in phosphorylation of a platelet protein (designated "peak 7" protein) of approximately 40,000 mol wt as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-150. A similar increase in phosphorylation was observed in a platelet protein (designated "peak 9" protein) of approximately 20,000 mol wt. The time for half-maximal phosphorylation of peak 7 and peak 9 protein was 10-14 s. The concentration of thrombin at half-maximal phosphorylation was 0.25 U/ml for both proteins. Prior incubation of platelets with dibutyryl cyclic adenosine 3',5'-monophosphate or prostaglandin E1 inhibited thrombin-induced peak 7 and peak 9 protein phosphorylation. The erythroagglutinating phytohemagglutinin of Phaseolus vulgaris, a non-proteolytic release-inducing agent, induced peak 7 and peak 9 protein phosphorylation. Thus, the characteristics of peak 7 and peak 9 protein phosphorylation are similar to those of the platelet release reaction, suggesting that the phosphorylation of these proteins may play a role in the platelet release reaction. When platelet sonicates or the supernatant fraction from platelet sonicates were incubated with [gamma-32P]ATP there was phosphorylation of both peak 7 and peak 9 proteins. This phosphorylation was unaffected by either added thrombin or adenosine 3',5'-cyclic monophosphate (cAMP) despite the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Thus, the thrombin-dependent phosphorylation depends upon intact platelets. When the supernatant fraction from platelet sonicates was fractionated by histone-Sepharose affinity chromatography, two distinct protein kinase enzymes were resolved, one a cAMP-dependent holoenzyme and the other a cAMP-independent enzyme. The isolated cAMP-dependent enzyme fraction catalyzed the cAMP-(but not thrombin-) stimulated phosphorylation of a protein that co-electrophoresed with peak 7 protein.
228 citations
TL;DR: It is suggested that avian vitellogenin is composed of two polypeptides, each of which has a molecular weight of approximately 240,000 and contains within it lipovitellin and two phosvitins.
220 citations
TL;DR: The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase.
202 citations
TL;DR: The constancy of the ratio of activities after isoelectric focusing and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids.
202 citations
TL;DR: A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating to the concentration of polymer -ligand and partition coefficient.
196 citations
TL;DR: The DNA-Sepharose is stable to high temperature, prolonged storage, and in the case of single-stranded DNA, can be washed with NaOH to destroy nuclease activity and to release any digested oligon nucleotides or mononucleotides.
Abstract: DNA has been covalently linked to insoluble matrices of agarose (Sepharose) in high yield using cyanogen bromide activation. Both double-stranded and single-stranded DNA have been coupled with yields up to 225 nmol/mg dry weight Sepharose or 3–8 μmol nucleotide phosphate/ml bed volume. The DNA-Sepharose has been used for (a) the affinity chromatography of various enzymes (Escherichia coli DNA polymerase I and RNA polymerase) from crude extracts or after initial purification steps, resulting in high yields and degrees of purification, and for (b) nucleic acid hybridization. The DNA-Sepharose is stable to high temperature, prolonged storage, and in the case of single-stranded DNA, can be washed with NaOH to destroy nuclease activity and to release any digested oligonucleotides or mononucleotides.
TL;DR: A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle and studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.
Abstract: A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.
TL;DR: Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography.
Abstract: Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.
TL;DR: The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions.
Abstract: An affinity column procedure is reported for purifying prolyl hydroxylase. The procedure is based on the affinity of the enzyme for its competitive polypeptide inhibitor, and involves affinity chromatography in a column containing poly(L-proline) of molecular weight 30000 linked to agarose, and the elution of the enzyme from the column with poly(L-proline) of molecular weight 5700. The enzyme is finally separated from this polyproline by gel filtration. The procedure was employed for purifying prolyl hydroxylase from an ammonium sulphate fraction of chick embryo extract. The recovery of enzyme activity varied in ten enzyme preparations from 50 to 82%, and the purified preparations synthesized from 59.3 to 91.5 mumol hydroxyproline per mg enzyme per h at 37 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The enzyme was pure when examined by polyacrylamide gel electrophoresis as a native protein or in the presence of sodium dodecyl sulphate, and the amino acid composition of the enzyme agreed with that reported previously with only minor exceptions. The molecular weight of the enzyme measured by equilibrium in an analytical ultracentrifuge was 240000, indicating that the enzyme had been isolated in the tetramer form.
TL;DR: TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties and its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone.
Abstract: This report concerns the purification and characterization of the testosterone-estradiol-binding globulin (TeBG) from human plasma. Cohn fraction IV was submitted sequentially to ammonium sulfate preciptation, affinity chromatography, gel filtration, and isoelectric focusing. The final product was homogeneous in polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone. Association constants (M-1) at 4 and 37degreesC were ascertained for three steroids: dihydrotestosterone; 2.4 x 10(9) and 0.99 x 10(9); testosterone, 1.1 x 10(9) and 0.35 x 10(9); estradiol, 0.60 x 10(9) and 0.22 x 10(9). TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties.
TL;DR: Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography and showed that the purified receptors contained both of the previously described 4 S progestersone binding components in near equal amounts.
TL;DR: The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes by preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns.
Abstract: The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.
TL;DR: Methods for purifying carbonic anhydrase isozymes by coupling sulfonamides to CM Sephadex using a water-soluble carbodiimide to form a peptide bond between the carboxyl and amino groups and azosulfonamide coupled gel had the advantages of a higher capacity and a red-colored product that enabled visual estimation of coupling efficiency.
TL;DR: The results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions.
Abstract: 1
Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein The yield of the purified enzyme was 25–45%
2
Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 87 nm Density gradient centrifugation in 01% Triton X-100 showed one peak of enzyme activity with an S4 value of 63S
3
Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 455, 468, 481, 498 and 518 Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point
4
After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 63 S, 102 S, 122 S, 142 S and 163 S At low ionic strength, higher aggregates were observed in addition to the other forms Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80000
5
These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions After removal of the detergent, forms with different molecular sizes are observed
TL;DR: After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off” of host cell protein synthesis is rapid.
TL;DR: The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotroropin binding to the solubILized receptor has been developed, and both preparations appear to exhibit a similar negatively cooperative relationship.
TL;DR: This simple, efficient method is used to prepare all the active components of the phytohemagglutinin family in large yield and high purity.
TL;DR: Protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions, and probably contains only one active site region.
Abstract: To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose.
From a partial tryptic digest of pure protein A at 30°C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A.
All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.
TL;DR: It is suggested that the increase of membrane fluidity is one of the common biochemical events in the earliest stage of lymphocytes transformation.
TL;DR: Under standard assay conditions, poly(A) polymerase activity was stimulated more than 10-fold by poly(C), but to small extent or not at all by other homopolyribonucleotides or natural RNA species unless they were first subjected to partial hydrolysis and alkaline phosphatase treatment.
TL;DR: It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinIn was obtained in crystalline form.
Abstract: A new and versatile method for linking biologically active ligands to a polyacrylamide matrix is reported. Active esters of acrylic acid (N-succinimicyl acrylate and N-phthalimidyl acrylate) were synthesized, then copolymerized with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active ester in the gel thus formed by various ligands containing aliphatic amino groups resulted in the formation of stable amid bonds between the ligands and the polyacrylamide gel. The affinity gel thus prepared has the following advantages: (i) resistance to chemical and microbiological degradation, (ii) ease of control of ligand level and higher levels of ligand possible, (iii) ease of control of porosity, and (iv) total displacement of the active ester under suitable conditions. Efficacy of this system was tested by preparation of 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside derivative polyacrylamide gel by the described method. It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinin was obtained in crystalline form. In addition, partial resolution of isolectins was obtained from the affinity gel witha pH gradient.
TL;DR: The present paper describes the characterization of the Thy-1 molecule from rat brain, which was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate, and was antigenically indistinguishable fromThy-1 from thymocytes that binds to lentil lectin.
Abstract: The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl alpha-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.
TL;DR: Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose to exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH.
TL;DR: A method for the preparation of homogeneous protein kinase regulatory subunit has been developed using adenosine-3′,5′-monophosphate (cAMP) affinity chromatography using N 6 -(2-Aminoethyl)-cAMP synthesized and covalently linked to CNBr-activated Sepharose 4B.
TL;DR: A close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix- bound ligand as a process quite similar to that occurring in solution.
Abstract: The elution of staphylococcal nuclease on thymidine 3'-(p-Sepharose-aminophenyl phosphate) 5'-phosphate (nucleotide ligand of nuclease covalently bound to Sepharose 4B) was studied in the presence of a variety of soluble nucleotide ligands. The elution volumes of nuclease vary proportionally with matrix-bound ligand concentration (at constant soluble ligand concentration), inversely with soluble ligand concentration (at constant matrix-bound ligand concentration), and inversely with dissociation constant of soluble ligand (at constant concentrations of soluble and matrix-bound ligand). The variation of elution volume was related to an expression which described the competition of soluble and matrix-bound ligand for nuclease binding. Using this expression, values for dissociation constants were derived for nucleotide ligands in both the soluble and bound form. The values for soluble ligand were found to correspond closely to those obtained by either equilibrium dialysis or kinetics of inhibition of nuclease activity. Furthermore, a close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix-bound ligand as a process quite similar to that occurring in solution.