Showing papers on "Affinity chromatography published in 1973"
TL;DR: Two enzymes isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride were active in releasing free fibers from filter-paper and indicated a homogeneous protein.
Abstract: 1
A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.
2
The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6B) and isoelectric focusing.
3
The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing.
4
Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis.
5
The molecular weights of the enzymes were 12500 and 50000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis.
6
The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 °C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%.
7
Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.
300 citations
TL;DR: The albumin-free plasma should be useful as a starting material for the isolation of many plasma proteins by classical procedures.
274 citations
TL;DR: Some basic features and potential uses of hydrophobic chromatography are described, and a homologous series of omega-aminoalkylagaroses that varied in the length of their hydrocarbon side chains was synthesized is synthesized.
Abstract: A homologous series of ω-aminoalkylagaroses [Sepharose-NH(CH2)nNH2] that varied in the length of their hydrocarbon side chains was synthesized. This family of agaroses was used for a new type of chromatography, in which retention of proteins is achieved mainly through lipophilic interactions between the hydrocarbon side chains on the agarose and accessible hydrophobic pockets in the protein.
When an extract of rabbit muscle was subjected to chromatography on these modified agaroses, the columns with short “arms” (n = 2 and n = 3) excluded glycogen synthetase (EC 2.4.1.11), but the enzyme was retained on δ-aminobutyl-agarose (n = 4), from which it could be eluted with a linear NaCl gradient. Higher members of this series (e.g., n = 6) bind the synthetase so tightly that it can be eluted only in a denatured form. A column of δ-aminobutyl-agarose, which retained the synthetase, excluded glycogen phosphorylase (EC 2.4.1.1), which in this column series and under the same conditions requires side chains 5-(or 6)-carbon-atoms long for retention. Therefore, it is possible to isolate glycogen synthetase by passage of muscle extract through δ-aminobutyl-agarose, then to extract phosphorylase by subjecting the excluded proteins to chromatography on ω-aminohexyl-agarose (n = 6). On a preparative scale, the synthetase (I form) was purified 25- to 50-fold in one step.
This paper describes some basic features and potential uses of hydrophobic chromatography. The relevance of the results presented here to the design and use of affinity chromatography columns is discussed.
209 citations
TL;DR: Human serum proteins were analyzed by crossed immuno-affinoelectrophoresis and affinity chromatography on concanavalin A bound to Sepharose and concordant results were obtained.
209 citations
TL;DR: A β-galactosidase was purified 600-fold from bovine testes by ammonium sulfate precipitation, acetone fractionation, and affinity chromatography on agarose substituted with terminal thio-β-Galactopyranosyl residues, and exhibited a high affinity for nitrophenylGalactosides.
187 citations
TL;DR: The findings suggest that binding of purified proteins for agarose substituted with 4-phenylbutylamine or with ϵ-aminocaproyl- d -tryptophan methyl ester results from the combined (and possibly mutually reinforcing) effects of hydrophobic and electrostatic forces.
185 citations
TL;DR: Results agree with kinetic studies, indicating that cyclic GMP-dependent protein kinases of arthropods have certain similarities to cyclic AMP- dependent protein kinase of vertebrates.
177 citations
TL;DR: The active form of ribonucleotide reductase consists of a 1:1 complex between proteins B1 and B2 of the type αα'β2, the type of which is strongly influenced by the presence of sucrose.
168 citations
163 citations
TL;DR: The most effective affinity adsorbents were prepared by attaching 17β-estradiol 17-hemisuccinate to agarose derivatives containing albumin or the branched copolymer of poly(l-lysine) ("backbone") and poly(dl-alanine) "side arms").
141 citations
TL;DR: The enzyme was shown to be a tetramer which has a molecular weight of about 230,000 and is composed of two different types of subunits, indicating that the structural integrity of the enzyme is maintained in part by either intrachain or interchain disulfide bonds.
TL;DR: Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin and that antireceptor antiserum will cause precipitation of more toxin- binding sites present in an initial extract than in purified receptor preparations.
Abstract: Rabbit antiserum against purified Electrophorus electricus acetylcholine receptor is studied using an immunoprecipitin assay to measure either antibody titer or concentration of toxin-binding sites in solubilized receptor preparations. This antiserum, unlike control serum, blocks the electrophysiological response of the electroplax to carbamylcholine. Toxin (α-neurotoxin, Naja naja) and several cholinergic ligands produce partial inhibition of the reaction of antiserum with purified acetylcholine receptor. Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin. In addition, evidence is presented that antireceptor antiserum will cause precipitation of more toxin-binding sites present in an initial extract than in purified receptor preparations.
TL;DR: The receptor for acetylcholine was partially purified by affinity chromatography of an extract in Triton X-100 of membrane fragments from electric tissue by reaction with the affinity-alkylating agent, [methyl-(3)H]4-(N-maleimido)-benzyltrimethylammonium iodide.
Abstract: The receptor for acetylcholine was partially purified by affinity chromatography of an extract in Triton X-100 of membrane fragments from electric tissue. The receptor was assayed, after its reduction with dithiothreitol, by reaction with the affinity-alkylating agent, [methyl-3H]4-(N-maleimido)-benzyltrimethylammonium iodide. Alternative labeling procedures, one useful for routine assay of picomole quantities of receptor and the other for labeling larger quantities of receptor, are described. The purified receptor specifically incorporated about 3 nmol of label per mg of protein. This incorporation was blocked by pretreatment of the receptor with Naja naja siamensis neurotoxin. The rate of the affinity reaction was similar to that found with membrane fragments and with intact electroplax. Furthermore, as in intact electroplax, [3H]4-(N-maleimido)-benzyltrimethylammonium iodide reacted 5000-fold faster with the reduced receptor than did [14C]N-ethylmaleimide. When purified receptor was labeled with [3H]4-(N-maleimido)-benzyltrimethylammonium iodide and subjected to electrophoresis on polyacrylamide gels in dodecyl sulfate and dithiothreitol, three major protein bands were observed. Only one of these, however, contained 3H activity; its mobility indicated a molecular weight of 40,000.
TL;DR: Human platelet surfaces were labeled with radioactive iodide utilizing lactoperoxidase and hydrogen peroxide generated by a glucose oxidase-glucose system and intense labeling of a 100,000 molecular weight membrane glycoprotein which was not altered by thrombin.
TL;DR: The isolated protein (20 µg) corrected vitamin B12 malabsorption when given to a patient with pernicious anemia in a standard Schilling test.
TL;DR: Alpha1-Fetoprotein present in fetal/newborn rat serum and in hepatoma-bearing human serum has been resolved into two molecular variants by concanavalin A-agarose affinity chromatography.
Journal Article•
TL;DR: The cytotoxic effector cell showed similar characteristics to monocytes with respect to size, surface adherence properties and binding affinity for immunoglobulin subclasses.
Abstract: Death of antibody-coated chicken erythrocytes (CRBC) mediated by nonphagocytic cells was studied. The distribution of effector cells in mouse spleen lymphoid populations after fractionation by velocity sedimentation on Ficoll gradients, isopycnic centrifugation on BSA gradients, and affinity chromatography on Sephadex-linked anti-Fab columns was followed. Cytotoxicity corresponded closely with the distribution of the non-phagocytic monocytes. The cytotoxic effector cell showed similar characteristics to monocytes with respect to size, surface adherence properties and binding affinity for immunoglobulin subclasses.
Journal Article•
TL;DR: Mouse interferon produced in L cells was subjected to affinity chromatography on Sepharose-bound anti-interferon globulin and adsorbed to remove antibodies against proteins derived from normal L cells, medium, and inducer virus preparation to permitted selective elimination of identified contaminant antigens.
Abstract: Mouse interferon produced in L cells was subjected to affinity chromatography on Sepharose-bound anti-interferon globulin. The hyperimmune anti-interferon serum was specifically adsorbed to remove antibodies against proteins derived from normal L cells, medium, and inducer virus preparation. The method permitted selective elimination of identified contaminant antigens from crude interferon preparations. Recovery of interferon was quantitative, and purification during this step was from 20- to 50-fold. Specific activities in peak fractions ranged from 1 to 2.7 × 108 National Institutes of Health reference units per mg protein. Interferons induced by irradiated Newcastle disease virus and poly I:C were purified to the same degree.
TL;DR: It is reported here that the serum of an immunized rabbit blocks the response of the isolated electroplax to bath applied carbamylcholine, and the same serum precipitates, in vitro, the receptor protein purified on the cholinergic column.
TL;DR: Bovine-serum albumin, known to have antipodal specificity in the binding of tryptophan, was selected as the affinity chromatographic matrix for the attemptedchromatographic resolution of DL-tryptophan.
Abstract: Bovine-serum albumin, known to have antipodal specificity in the binding of tryptophan, was selected as the affinity chromatographic matrix for the attempted chromatographic resolution of DL-tryptophan. Complete resolution was accomplished when Dl-tryptophan was chromatographed on bovine-serum albuminsuccinoylaminoethyl-Sepharose.
TL;DR: Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration and the values of the inhibition constant, K i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer forThrombin than for trypsIn.
TL;DR: The agglutinating reaction with red cells was inhibited by sugars containing galactose residues, and the purified lectin gave a single peak on Sephadex G-200 gel filtration with an elution volume corresponding to a molecular weight of 120 000.
TL;DR: An improved procedure, utilizing affinity chromatography in the final step, has been devised for the purification of the NADP-specific glutamate dehydrogenase of Neurospora crassa, and present evidence indicates that the reaction with N-ethylmaleimide probably involves the same e-amino group as in the Reaction with pyridoxal 5'-phosphate.
TL;DR: The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined andSepharose was found to be superior.
Abstract: 1. The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined. Sepharose was found to be superior. 2. The selective capacities of the columns were measured by frontal analysis and are discussed in relation to the nucleotide contents. 3. The effect of various concentrations of enzyme and of non-specific protein on the performance of the affinity columns, and the effects of equilibration time, flow rate, sample volume and dilution of the nucleotide were examined. 4. The effect of interposing polymethylene and polyglycine extension arms between the matrix backbone and the nucleotide was investigated for several cofactor-dependent enzymes. Maximum binding was observed with an extension arm 0.8–1nm long.
TL;DR: Efforts were made to isolate fatty acid binding regions of the bovine albumin molecule after tryptic digestion of albumin which was bound to fatty acid-agarose, and some implications on the properties of the fatty acids-binding sites on albumin have been offered.
TL;DR: The purified factor V-activating enzyme from Russell's viper venom converts factor V to a form which binds to a prothrombin-Sepharose affinity column, which is quantitatively eluted from the affinity column by increasing the ionic strength of the buffer.
Journal Article•
TL;DR: The purified agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes.
TL;DR: Aminoacyl-tRNA synthetases adsorbed on phosphocellulose can be specifically eluted with purified tRNA with the advantage of the use of enzyme-ligand affinity without fixation of the ligand to a polymer support.
Abstract: Aminoacyl-tRNA synthetases adsorbed on phosphocellulose can be specifically eluted with purified tRNA. This method we call affinity elution in analogy to affinity chromatography. The advantage of the method is the use of enzyme-ligand affinity without fixation of the ligand to a polymer support. As an example, purification of phenylalanyl-tRNA synthetase, valyl-tRNA synthetase, seryl-tRNA synthetase and isoleucyl-tRNA synthetase from baker's yeast is described. The first two aminoacyl-tRNA synthetases were obtained as apparently homogeneous proteins with purification factors 537 and 246, respectively.