Showing papers on "Affinity chromatography published in 1974"
TL;DR: In this article, a new method for preparing adsorbents for biospecific affinity chromatography is described, and the optimal conditions for the activation and subsequent coupling of proteins, peptides and aliphatic and aromatic amines have been evaluated.
641 citations
TL;DR: The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions.
Abstract: Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3145) In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000 (c) It has an amino-acid composition closely similar to that of actin (d) The peptide maps of the two proteins are nearly identical (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions
604 citations
TL;DR: The purification, by affinity chromatography, of an hepatic protein which retains the characteristic binding properties associated with the membranes is described, which indicates a high degree of aggregation in the final, water-soluble preparation.
555 citations
TL;DR: The pure preparation showed both progressive antithrombin activity and heparin cofactor activity, which presumably consists of a single polypeptide chain without disulphide bridges.
420 citations
TL;DR: It is suggested that Bandeirae simplicifolia lectin, the first α-d-galactopyranosyl binding lectin (anti-B lectin) to be purified and characterized, will be valuable not only as a serological reagent, but in studies of polysaccharides, glycoproteins, and cell membrane structure.
351 citations
TL;DR: It is demonstrated that affinity chromatography of human erythrocyte membranes on lectin-Sepharose columns results in the separation of two distinct classes of glycoproteins with different carbohydrate compositions and different lectin binding properties.
255 citations
TL;DR: To establish a procedure for the purification of hypothetical protein, X, the primary required reagent is specific anti-X antibody, covalently bound to an insoluble matrix, and one must establish chromatographic conditions that permit only specific binding of protein X to a column of the immunoadsorbent.
Abstract: Publisher Summary In principle, protein antigen X, contained in a mixture of many other proteins, will bind selectively to insolubilized anti-X immunoglobulin (IgG). The other proteins for which the antibody has no affinity can be washed away and discarded. Thereafter, protein X is eluted from the adsorbent under conditions that disrupt the immune complex. The procedure has become widely applicable, having been developed for the purification of a variety of enzymes and other proteins. To establish such a procedure for the purification of hypothetical protein, X, the primary required reagent is specific anti-X antibody, covalently bound to an insoluble matrix. In addition, one must establish chromatographic conditions that permit only specific binding of protein X to a column of the immunoadsorbent. Thus, a similar column containing covalently linked nonimmune IgG should bind little or no protein X. Moreover, under ideal conditions, X will be the only macromolecule binding to the specific immunoadsorbent. Because of strong antigen binding, elution conditions which, of necessity, may not be as gentle as in ionic adsorption chromatography, and must be both sufficiently drastic to remove the protein completely and sufficiently gentle to permit its isolation in biologically active form.
241 citations
TL;DR: The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx.
Abstract: The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx. 300-fold from Triton X-100 extracts of electroplax membrane fragments. The receptor protein was first adsorbed from crude extracts on an affinity column containing 1,3-bis(tri-ethylammonium ethoxy)-4-iodoacetamidobenzene diiodide, an analogue of flaxedil, linked to Sepharose 2B, then eluted with a solution of flaxedil and concentrated. The receptor protein was further purified by centrifugation on sucrose gradients containing 1 Triton X-100, 1% Emulphogene, or 0.5% sodium cholate. If desired, detergent could be removed after centrifugation in the presence of sodium cholate by gel filtration on Sephadex G-75. At all stages the receptor was assayed by incubation with α-[3H]toxin from Naja nigricollis followed by isolation of the toxin · receptor complex by filtration on Millipore filters at low detergent concentration. The best preparations bound 1 mol toxin per 150000 g protein.
In the presence of non-denaturing detergents the preparations gave a single major band for both protein and activity upon polyacrylamide disc gel electrophoresis or sucrose gradient centrifugation. The receptor protein appears essentially homogeneous in the electron microscope after negative staining with uranyl acetate. Upon gel electrophoresis in the presence of dodecylsulphate, there were two major bands corresponding to molecular weights of 43000 and 48000.
The ultraviolet-absorption spectrum and amino-acid composition of the purified receptor protein are typical of those of globular, water-soluble proteins in general and do not reflect the hydrophobic character of the macromolecule. Furthermore, the purified protein interacts with concanavalin A and other plant agglutinins indicating that it contains a carbohydrate moiety.
The purified receptor protein carries a single class of binding sites for [3H]decamethonium (Kd= 0.02 μM) at ligand concentrations up to 1 μM and for acetylcholine (Kd= 0.06 μM) at up to 1 μM. No evidence for cooperativity in binding of cholinergic effectors to the receptor protein, either purified or in crude extracts, has been detected and all effectors tested inhibit the binding of [3H]decamethonium in a competitive manner. The affinity of agonists for the solubilized receptor protein is approx. 20-fold greater than for the membrane-bound protein.
228 citations
TL;DR: It is suggested that it is feasible to utilize the soluble receptor for the development of a very sensitive radioreceptor assay for prolactin and, further, to obtain a highly purified receptor in sufficient quantity to facilitate studies on its physiological, biochemical, and immunological properties.
209 citations
TL;DR: Examination of the tryptic digests of the subunits showed that the α subunit is not a fragment of the β subunit produced by a specific proteolytic cleavage, and pea lectin purified by affinity chromatography is a mixture of two closely related proteins which have the same subunit structure but which differ on a charge basis.
161 citations
TL;DR: Lipoprotein lipase was purified from acetone powders of pig adipose tissue and exhibited a complete dependence on the presence of serum for hydrolysis of triolein emulsions stabilized with gum arabic, as well as three very low density lipoprotein apoproteins studied.
TL;DR: It is concluded that aortic lysyl oxidase is a copper metalloenzyme that catalyzed the formation of tritiated water from embryonic aortas grown in organ culture.
TL;DR: In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.
Abstract: Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100-lysozyme-d-glucose system in acetate-chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m(-1) and 1.2x10(6)m(-1) are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 10(5) glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.
TL;DR: Application of the procedure to the striated muscle of rabbit has indicated that there are at least three forms of troponin I present in this tissue, each form being specific for the type of muscle from which it is isolated.
TL;DR: A procedure is demonstrated for the direct binding of hydroxyl group-containing ligands to Sepharose 6 B which has been previously activated by a bir‘unctional epoxide for biospecific affinity chromatography of sweet potato /3-amylase.
TL;DR: Rat erythrocytes contain a 3β-hydroxysteroid dehydrogenase, which converts 5 α-dihydrotestosterone reversibly into 5α-androstane-3β,17β-diol and both activities are probably expressions of a single enzyme.
TL;DR: In this article, the same proteins were not adsorbed by the corresponding alkyl hydrazide derivatives of agarose, but were bound strongly to tyramine-agarose with partial inactivation of the enzyme Leucine aminopeptidase.
TL;DR: Chitin, the naturally occurring polymer of N-acetyl-glucosamine, has been used as a ligand matrix for affinity chromatography of wheat germ agglutinin in a new purification method which is well suited for large scale preparations.
TL;DR: The major viral glycoprotein of Friend leukemia virus was isolated by a procedure comprising density gradient centrifugation, Con A affinity chromatography, and Sephadex gel filtration and found to be pure by physicochemical and seroimmunological methods.
TL;DR: In this article, the affinity chromatographic separation procedure was tested for its utility in quantifying the affinity of proteins to both insolubilized ligand and corresponding competing soluble ligand.
Abstract: The affinity chromatographic separation procedure was tested for its utility in quantitating the affinity of proteins to both insolubilized ligand and corresponding competing soluble ligand. An expression has been derived that (1) allows the determination of binding constants for the interaction of a soluble protein species with an affinity chromatography matrix involving active site binding, (2) yields binding constants for the interaction of the protein with soluble ligands that can compete with the insoluble matrix for binding to the active site of the protein, and (3) utilizes readily obtainable parameters from conventional chromatography. This expression has been applied to the quantitation of the binding of staphylococcal nuclease to thymidine-5′-phosphate-3′-amino-phenylphosphate-Sepharose in competition with thymidine-3′,5′-bisphosphate.
TL;DR: The bleomycin-inactivating enzyme of rat liver was separated from a known aminopeptidase B by Affinity chromatography of the extract on Sepharose 4 B-lysinamide gave 25-fold purified enzyme, but further purification vas not successful because of instability of the enzyme.
Abstract: Affinity chromatography of a bleomycin-inactivating enzyme was studied on Sepharose 4 B-bleomycin A5 and Sepharose 4 B-lysinamide. Mouse liver was homogenized, and the enzyme extracted by ammonium sulfate precipitation and Sephadex G-200 chromatography. Affinity chromatography of the extract on Sepharose 4 B-lysinamide gave 25-fold purified enzyme, Further purification vas not successful because of instability of the enzyme. It hydrolyzed L-lysinamide, L-lysyl-β-naphthyl-amide, L-arginy1-β-naphthylamide, L-lysyl-L-lysine and Iysyl-bradykinin, but not leucinamide or L-leucyl-β-naphthylamide. Hydrolysis of L-lysyl-β-naphthylamide and L-arginyl-β-naphthylamide by the enzyme was competitively inhibited by bleomycin B2. The bleomycin-inactivating enzyme of rat liver was separated from a known aminopeptidase B.
TL;DR: It appears that insulin and glucagon can be degraded by the same enzyme which is found in the soluble fraction of rat skeletal muscle homogenate.
Abstract: An insulin-degrading enzyme has been previously isolated from rat skeletal muscle and highly purified by a series of procedures including affinity chromatography on insulin-agarose. Glucagon-degrading activity was also found in each of the purification steps including the final highly purified material. Insulin- and glucagon-degrading activities were associated with the same band on polyacrylamide gel and both activities were obtained from affinity chromatography on glucagon-agarose. Insulin- and glucagon-degrading activities could not be differentiated by the effect of sulfhydryl reagents, pH, or by heat inactiva-tion. Insulin served as a competitive inhibitor of glucagon degradation (Ki = 1.5 × 10 −8 M) and glucagon was a competitive inhibitor of insulin degradation (Ki = 5.3 × 10 −6 M). On the basis of these studies it appears that insulin and glucagon can be degraded by the same enzyme which is found in the soluble fraction of rat skeletal muscle homogenate.
TL;DR: The purified enzyme had the highest specific activity so far reported for a human serum-stimulated lipase, and was active against tributyrylglycerol also in the absence of exogenous serum factors.
TL;DR: The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin-agarose resin and shows a difference in polypeptide composition, shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.
Abstract: The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin-agarose resin. After subsequent separation from an inactive higher-molecular-weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5: this proteolytic activity is completely inhibited by the acid-proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.
TL;DR: The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and at least one elastase from activated extracts of human pancreas has been accomplished by a combination of affinity chromatography and conventional ion-exchange chromatography on SE-Sephadex.
Abstract: The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and at least one elastase from activated extracts of human pancreas has been accomplished by a combination of affinity chromatography and conventional ion-exchange chromatography on SE-Sephadex. The enzymes thus obtained were homogeneous by several criteria: gel electrophoresis, equilibrium sedimentation and immunological assay. The molecular weight and amino-acid composition of each preparation was determined and compared with those of other mammalian enzymes. The two trypsins showed only partial immunological identity while the two chymotrypsins were identical immunologically. The data raises the question of the number of the zymogens present in the human pancreas.
In addition to the proteolytic enzymes, four protein inhibitors of proteolytic enzymes were also isolated from human pancreas. The procedure consisted of several steps that included gel filtration, affinity chromatography and isoelectric focusing. The two major components which accounted for most of the trypsin inhibitory activity were characterized and were apparently of the Kazal type. The more basic human pancreatic inhibitor had a pI of 8.7 and contained 48 amino acids with a molecular weight of 5200. The more acidic inhibitor had a pI of 6.5 and contained 39 amino acids with a molecular weight of 4300. Both inhibitors were capable of inhibiting bovine and two human trypsins. They did not inhibit two human chymotrypsins but had a weak capacity to inhibit bovine chymotrypsin.
TL;DR: Human β-glucuronidase has been isolated from four sources by affinity chromatography on a heterologous antibody resin and enzyme preparations from blood platelets had far greater uptake and corrective activity than enzymes from liver, placenta, or urine.
TL;DR: The HN protein can be displaced from the fetuin by the neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid, which indicates that a site on the HNprotein binds specifically to the neuraminationic acid moieties of the fetulin.
TL;DR: Affinity gels with group rather than compound specificity have the potential advantage of being useful for the isolation of many compounds belonging to a particular group, since highly selective compound elution is still possible with substrates, cofactors, or inhibitors.
Abstract: Biospecific affinity chromatography has been shown to be a powerful method for the isolation of biological compounds1,2. While high specificity of the immobilized ligand for the compound of interest generally results in high purification factors and simple isolation protocols, it usually necessitates the often laborious synthesis of a special affinity adsorbent. Affinity gels with group rather than compound specificity therefore have the potential advantage of being useful for the isolation of many compounds belonging to a particular group. The resolving po!’.er of these gels need not be less than the more compound specific gels, since highly selective compound elution is still possible with substrates, cofactors, or inhibitors.