Showing papers on "Affinity chromatography published in 1972"
TL;DR: The isolation of protein A from S. aureus and of IgG from human serum by affinity chromatography is described and several uses for protein A bound to a solid support as an immunosorbent will be discussed.
582 citations
437 citations
TL;DR: This chapter focuses on vertebrate lysozymes, particularly hen egg-white (HEW) lysozyme, which is small and basic, and it separates well on weak acid resins, like Amberlite XE-64 or Bio-Rex 70 or carboxymethyl cellulose and on calcium phosphate gel.
Abstract: Publisher Summary This chapter focuses on vertebrate lysozymes, particularly hen egg-white (HEW) lysozyme. Lysozymes of other types generally are distinguishable from HEW lysozyme in having higher molecular weights and somewhat different enzymic activities. Lysozyme is small and basic, and it separates well on weak acid resins, like Amberlite XE-64 or Bio-Rex 70 or carboxymethyl cellulose and on calcium phosphate gel. Affinity chromatography of lysozyme has been carried out using dispersed chitin or carboxymethyl (CM)-chitin. The conditions required for elution from chitin columns suggest that there are two classes of adsorbent sites that are differently affected by pH and ionic strength in their interaction with the enzyme. The preparation of lysozyme from diverse animal sources is generally achieved in four main steps, which include (1) the preparation of a lysozyme-rich extract, (2) chromatography on CM-cellulose, (3) filtration on Sephadex G-25, and (4) ion exchange chromatography on Amberlite CG-50 at 20° C with a 0.2 M phosphate buffer. The chromatography is sensitive to the pH and small variation of no more than 0.1 pH unit can involve complete retention or exclusion.
317 citations
275 citations
TL;DR: On the basis of the properties of the adsorbents, it has been possible to purify the galactosyltransferase from bovine milk to constant specific activity soley by affinity chromatography.
258 citations
TL;DR: It was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%.
Abstract: Messenger RNA from polysomes of KB-cells was isolated by affinity chromatography on columns of polyuridylic acid covalently linked to Sepharose. The mRNA molecules were retained by the resin apparently via their poly(A) segments by base pairing to the poly(U). Ribosomal RNA and transfer RNA were not retained by the columns and were thus removed from the mRNA. The mRNA was recovered to an extent of 90% and apparently in intact form.
This method allows studies of mRNA resulting from unabated synthesis and was used here in studies of the size distribution of different classes of cytoplasmic poly(A)-containing RNA. The presence of poly(A)-containing RNA in the non-polysomal fractions of the cytoplasm was demonstrated. This putative mRNA was shown to constitute about 30% of the total cytoplasmic poly(A)-containing RNA. Also by using the poly (U)-Sepharose technique it was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%. This low concentration of the drug was previously though to effect only ribosomal RNA synthesis.
207 citations
TL;DR: Relatively simple and rapid procedures are described for the large-scale preparation of liver membranes that contain virtually all of the high affinity insulin-binding activity of liver homogenates, and the receptor macro-molecule approaches theoretical purity on the basis of its specific activity.
Abstract: Relatively simple and rapid procedures are described for the large-scale preparation of liver membranes that contain virtually all of the high affinity insulin-binding activity of liver homogenates. The presumed insulin recepotr, which is extracted from these membranes in soluble form with Triton X-100, can be further purified by ammonium sulfate fractionation (3-fold purification) or by diethylaminoethyl-cellulose chromatography (60-fold purification). Several insulin-agarose derivatives have been synthesized that can efficiently extract the insulin-binding protein from the detergent extracts of the membranes. The receptor macro-molecule can be eluted from the affinity columns in high (50-80%) yield by use of urea-containing buffers of moderately low pH. The receptor, thus purified by small-scale affinity chromatography experiments, approaches theoretical purity on the basis of its specific activity. This protein is purified about 250,000-fold from the liver homogenate by detergent extraction and affinity chromatography.
204 citations
TL;DR: Agarose gels containing immobilized single-stranded circular DNA from phage fd or denatured calf thymus DNA were investigated for their use in the affinity chromatography of DNA-binding enzymes.
Abstract: Agarose gels containing immobilized single-stranded circular DNA from phage fd or denatured calf thymus DNA were investigated for their use in the affinity chromatography of DNA-binding enzymes. The DNA content of gel fragments is stable under the conventional conditions of enzyme purification. Single-stranded DNA-agarose columns have a high capacity to bind DNA-specific proteins. They were used to differentiate between similar enzymatic activities in DNA-free extracts from Escherichia coli. Preparative purification is described for the following enzymes: E. coli DNA polymerase I, DNA polymerase II, RNA polymerase, exonuclease III and T4 polynucleotide kinase. Enzyme purification was as high as 200-fold, recovery of enzymatic activity was 75–100%.
191 citations
TL;DR: The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydration to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m.
Abstract: 1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.
174 citations
TL;DR: An affinity column is described which gives, in a single step and with a particularly high yield, an approx.
165 citations
TL;DR: The enzyme had the characteristics of a lipoprotein lipase, i.e. its activity against emulsified long chain triglyceride was stimulated more than 20-fold by addition of suitable amounts of serum to the assay system and the activity was almost completely inhibited by 1 m NaCl.
TL;DR: Isolation of specific receptors for hormones, drugs and other biologically active molecules are frequently located at the cell surface, but isolation of these receptors has been impeded by the insolubility of cell membranes in the more orthodox biochemical solvents.
Abstract: CURRENT interest in the structure of cell plasma membranes arises partly from demonstrations that specific receptors for hormones (for example, insulin and glucagon1), drugs and other biologically active molecules (for example, Phaseolus vulgaris phytohaemagglutinin2, concanavalin A3 and acetylcholine4,5) are frequently located at the cell surface. Isolation of these receptors has, however, been impeded by the insolubility of cell membranes in the more orthodox biochemical solvents, whereas more drastic solubilization procedures have usually destroyed biological activity6.
TL;DR: Phytoagglutinine, welche Galaktose spezifisch binden, wurden mit Chromatographie an Sepharose gereinigt und durch Gelfiltration mittels Bio-gel getrennt.
Abstract: Phytoagglutinine, welche Galaktose spezifisch binden, wurden mit Chromatographie an Sepharose gereinigt. Zwei Fraktionen ausRicinus communis-Agglitomom wiurden durch Gelfiltration mittels Bio-gel getrennt: die fruh eluierte Fraktion zeigte starke Agglutinationsaktivitat und schwache Toxizitat, wahrend die spater eluierte Fraktion eine schwache Agglutinatinsaktivitat bei starker Toxizitat zeigt.
TL;DR: This property has now been utlized by us for the purification of SBA on a column made of a conjugate of Sepharose and N-e-aminocaproyl-/3-D-galactopy ranosylamine (SAG), a lectin isolated from soybean oil meal.
TL;DR: A single enzyme that proteolytically degrades insulin was isolated from rat skeletal muscle and purified 1000-fold by a series of steps, including affinity chromatography on insulin bound to agarose at the NH(2)-terminal phenylalanine of the B chain.
Abstract: A single enzyme that proteolytically degrades insulin was isolated from rat skeletal muscle. This enzyme was purified 1000-fold by a series of steps, including affinity chromatography on insulin bound to agarose at the NH2-terminal phenylalanine of the B chain. Insulin linked to agarose at the B-29 lysine residue did not bind the enzyme and, therefore, was not suitable for purification procedures. Insulin linked at the phenylalanine residue was a substrate for the enzyme and was degraded by it; insulin attached to agarose at the lysine residue was not degraded by the enzyme. The purified enzyme preparation yielded one major band on polyacrylamide gel electrophoresis, and elution of this area of the gel yielded insulin-degrading activity. The purified enzyme degraded insulin but not proinsulin, with a Km for insulin of 22 nM and a Ki for proinsulin of 40 nM. The enzyme is sulfhydryl-dependent, with a physiological pH optimum.
TL;DR: Model studies on lactate dehydrogenase (LDH) demonstrate that the effectiveness of affinity chromatography as a purification tool for certain types of multi-substrate enzyme may be greatly increased by taking advantage of kinetic characteristics.
TL;DR: A method of affinity chromatography which is a potent tool for isolation of the trace vitamin B12 binding proteins has been developed as mentioned in this paper, which was prepared by partial acid hydrolysis (0.4 n HCl, 64 hours, room temperature) of the amide groups of the unsubstituted propionamide side chains of the corrin ring of vitamin b12.
TL;DR: Analysis of transcobalamin II for carbohydrate content using gas-liquid chromatography and amino sugar analysis by the amino acid analyzer suggest that this trace plasma protein is not a glycoprotein.
TL;DR: A careful series of protein determinations was carried out to establish the ultraviolet extinction coefficient of the purified enzyme, which appears to be the distance the inhibitor is extended from the gel matrix by a chain of bridging atoms which form an attachment arm.
TL;DR: Highly purified acetylcholinesterase was obtained if affinity chromatography was preceded by controlled tryptic digestion or prolonged autolysis causing conversion of the enzyme to an 11-S form which does not aggregate at low ionic strength.
TL;DR: A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility was solubilized from a microsomal fraction of canine ventricular myocardium and purified 500 to 800-fold with conjugates of norepinephrine linked to agarose beads.
Abstract: A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified beta-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 x 10(5) liters/mol (as compared to two association constants, 10(7) and 10(6) liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for beta-adrenergic drugs and antagonists.
TL;DR: An acetylcholine receptor (AChR) affinity resin was prepared by covalently linking [N-(ϵ-aminohexanoyl)-3-aminopropyl]trimethyl ammonium bromide hydrobromide to agarose to produce a partially purified membrane protein preparation that displayed α-bungarotoxin binding activity.
TL;DR: An affinity chromatography technique was developed to isolate the five penicillin-binding components present in Bacillus subtilis membranes and enabled this enzyme to be obtained from the membrane in pure form in a single step with 50% overall recovery of enzymatic activity.
Abstract: An affinity chromatography technique was developed to isolate the five penicillin-binding components present in Bacillus subtilis membranes. The proteins were solubilized by the detergent Nonidet P-40, bound covalently to penicillin-substituted Sepharose, and subsequently eluted from the matrix with neutral hydroxylamine, which cleaves the penicilloyl-enzyme bond. Penicillin binding-component V, the D-alanine carboxypeptidase, makes up 1% of the total membrane protein. A modification of the above procedure enabled this enzyme to be obtained from the membrane in pure form in a single step with 50% overall recovery of enzymatic activity.
TL;DR: It is concluded that heparin stabilizes rather than stimulates the enzyme; furthermore, the effect of inhibitors is partly or completely abolished.
TL;DR: The vitamin B12-binding protein isolated from human granulocytes derived from patients with chronic granulocytic leukemia was purified 9,860-fold with a yield of over 90% and was homogeneous based on polyacrylamide disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate polyacylamide gel electrhoresis.
TL;DR: A similar procedure is described which produces, after purification by affinity chromatography on a column of bovine neurophysins immobilized on Agarose, a highly radioactive compound which exhibits all the biological and biochemical properties of the native hormone.
TL;DR: This binding protein has now been further purified by affinity chromatography by means of folic acid covalently bound to agarose gel through 1,6_diaminohexane.
TL;DR: It is indicated that methotrexate-resistant cells accumulate large quantities of a very similar, if not identical, reductase, due at least in part to an increased rate of enzyme synthesis.
TL;DR: The isolation of yeast phenylalanyl-tRNA synthetase in a completely pure state is described by an affinity chromatography technique, and this enzyme has been isolated in a homogeneous state before.