Showing papers on "Affinity chromatography published in 1981"
TL;DR: A method is described for the affinity purification of antibodies using protein samples that have been electrophoretically transferred to diazotized paper and antibodies were isolated that specifically bound only to tubulin on blots and that stained microtubule networks in cells.
789 citations
TL;DR: A cell surface glycoprotein antigen with an apparent molecular weight of about 100,000 that is selectively expressed on proliferating cells was purified from deoxycholate-solubilized membranes of a cultured human leukemic thymus-derived (T) cell line by affinity chromatography on a monoclonal antibody-Sepharose column and it was found to contain antibodies against a serum component that bound tightly to cultured cells.
Abstract: A cell surface glycoprotein antigen with an apparent molecular weight of about 100,000 that is selectively expressed on proliferating cells was purified from deoxycholate-solubilized membranes of a cultured human leukemic thymus-derived (T) cell line by affinity chromatography on a monoclonal antibody-Sepharose column. A conventional xenoantiserum prepared by immunization with the affinity-purified glycoprotein was found to contain antibodies against a serum component that bound tightly to cultured cells. This molecule was shown to be specifically associated with the cell surface glycoprotein purified by immunoprecipitation from lysates of cells. We have identified the serum component as transferrin and conclude that the membrane glycoprotein is the cell surface transferrin receptor.
665 citations
TL;DR: Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes, but this chapter presents a procedure for the preparation of glutathioneTransferase from human placenta.
Abstract: Publisher Summary Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes. However, this chapter presents a procedure for the preparation of glutathione transferase from human placenta. Enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione and l-chloro-2,4-dinitrobenzene. Purification procedure involve the extraction from the human placenta; the supernatant fraction from Step 1 is chromatographed on a column (12.5 x 80 cm) of Sephadex G-25 and the pooled fractions from Step 2 are applied to a column (9 x 13 cm) of DEAE-cellulose equilibrated with l0 mM Tris-HCl at pH 7.8. The pooled effluent from Step 3 is dialyzed overnight against 3 x 10 liter of 10 mM sodium phosphate buffer; then affinity chromatography is carried on S-hexylglutathione coupled to epoxy-activated sepharose 6B and the pooled fractions from Step 5 are charged onto a column of Sephadex G-75 that is equilibrated and eluted with 10 mM sodium phosphate at pH 6.7, containing 0.1 mM dithioerythritol. The active fractions of effluent are pooled.
549 citations
TL;DR: Results show that the interaction of fibronectin with cells is restricted to a defined portion of the molecule and is independent of the direct involvement of the known affinities toward other macromolecules.
437 citations
TL;DR: Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture, which blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinases, but not thermolysin or bacterial collagenase.
Abstract: 1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.
327 citations
TL;DR: Evidence is provided that increased nonenzymatic glycosylation is occurring in a tissue where physiological, morphological, and clinical degeneration characteristically develop as a result of diabetes mellitus.
Abstract: A new affinity chromatography system that selectively retains glycosylated amino acids has been utilized to determine the amount of nonenzymatic glycosylation present in peripheral nerve from diabetic and control rats and dogs. The mean value for glycosylated amino acids in diabetic rats was 2.8 times greater than the mean value in normal rats (P less than 0.001). In diabetic dogs, mean values were 2.15 times greater than normal values (P less than 0.05). Amino acid analysis of reduced, glycosylated amino acids previously isolated by affinity chromatography showed that glycosylated lysine and its hydrolysis rearrangement products were the major borohydride-reducible adduct present. In addition, another glycosylated product was noted to be present in major proportions. This radioactive product did not chromatograph with any of the available glycosylated amino acid standards. The finding that diabetes results in a nearly 3-fold increase of peripheral nerve glycosylation is consistent with a number of previous investigations in which glycosylation was measured in hemoglobin, serum albumin, and urinary amino acids and peptides from diabetics and normals. The results reported here provide evidence that increased nonenzymatic glycosylation is occurring in a tissue where physiological, morphological, and clinical degeneration characteristically develop as a result of diabetes mellitus.
223 citations
TL;DR: N6-(6-aminohexyl)-AMP coupled to diol-silica completely separated a mixture of albumin, lactate dehydrogenase and alcohol dehydrogenases in less than 15 min when the technique of high performance liquid affinity chromatography was employed.
223 citations
TL;DR: This chapter presents the procedure for purification and assaying of mammalian collagenases, which proteinases acting at neutral pH to cleave the native helix of the interstitial collagens.
Abstract: Publisher Summary This chapter presents the procedure for purification and assaying of mammalian collagenases, which proteinases acting at neutral pH to cleave the native helix of the interstitial collagens. Collagenase can be purified from the culture media of a variety of tissues or cells from different species. The purification steps include: ammonium sulfate precipitation; gel filtration with ultrogel AcA 44; DEAE-cellulose chromatography; and zinc chelate affinity chromatography. At the present time, only assay systems employing collagen itself as a substrate are suitable for the unambiguous detection of specific collagenases. Type I collagen is normally used as the substrate for the assay of collagenase and is prepared in soluble form from skin and tendon. Soluble collagen can be reconstituted into insoluble fibrils by incubating at 25-37°C at neutral pH and this provides the basis for the most widely used collagenase assays. Radiolabeled collagen fibrils are incubated with the enzyme and collagen degradation followed by the assay of radioactivity solubilized during the assay period.
210 citations
TL;DR: The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins with wheat germ agglutinin (WGA) were investigated by using affinity chromatography on a WGA-Sepharose column and it was revealed that both the N,N'-diacetylchitobiose moiety and the beta-N-acetylglucosaminyl residue linked to C-4 of thebeta-linked mann
Abstract: The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins with wheat germ agglutinin (WGA) were investigated by using affinity chromatography on a WGA-Sepharose column. So-called hybrid-type glycopeptides obtained from ovalbumin [Yamashita, K., Tachibana, Y., & Kobata, A. (1978) J. Biol. Chem. 253, 3862--3869] were found to have high affinity for WGA--Sepharose, whereas high mannose-type and complex-type glycopeptides were shown to have low affinity. The elution profiles of various glycopeptides modified by glycosidase treatment, Smith periodate degradation, acetolysis, and hydrazinolysis showed that the GlcNAcbeta 1--4Manbeta 1--4GlcNAc beta 1--4GlcNAc-Asn structure was essential for the binding of glycopeptides to a WGA-Sepharose column. Thus, it was revealed that both the N,N'-diacetylchitobiose moiety and the beta-N-acetylglucosaminyl residue linked to C-4 of the beta-linked mannose residue contributed to the interaction of the glycopeptide with WGA-Sepharose. The substitution at C-6 of the innermost beta-N-acetylglucosaminyl residue by an alpha-fucosyl residue or at C-6 of the beta-linked mannose residue by another mannose residue in the above structure reduced the affinity of glycopeptides for the column.
209 citations
TL;DR: Eukaryotic mRNA cap binding proteins were purified from ribosomal salt wash in the presence of protease inhibitors by sucrose gradient sedimentation and m7GDP-Sepharose affinity chromatography and stimulated capped mRNA translation in extracts of uninfected HeLa cells but did not restore capped mRNA function in extracts prepared from poliovirus-infected cells.
204 citations
TL;DR: Tissue factor (tissue thromboplastin, factor III), an initiator of coagulation, has been purified 142,000-fold to homogeneity from bovine brain, demonstrating that the protein observed on the gels is tissue factor.
TL;DR: It is shown here that affinity chromatography with these antibodies is effective for the small-scale isolation of some PT-proteins, including p120, the transforming protein of Abelson murine leukaemia virus, and several unidentified proteins from Rous sarcoma virus -transformed mouse fibroblasts.
Abstract: Tyrosine-specific protein kinases seem to be involved critically in cellular transformation by tumour viruses1–4, and may also be involved in the cellular response to epidermal growth factor5. To facilitate the identification and isolation of phosphotyrosine-containing proteins (PT-proteins) we have sought to develop as a general reagent anti-O-phosphotyrosine antibodies. We show here that affinity chromatography with these antibodies is effective for the small-scale isolation of some PT-proteins, including p120, the transforming protein of Abelson murine leukaemia virus, and several unidentified proteins from Rous sarcoma virus (RSV)-transformed mouse fibroblasts.
TL;DR: The acceptor substrate specificity of the transferase(s) and structural characterization of the reaction products indicate that the enzyme(s), formed the Fuc alpha 1 goes to 4GlcNAc, Fuc α 2 goes to 3Glc NAc, and Fuc 1 goes on to 3glc linkages with oligosaccharide acceptors containing the nonreducing terminal sequences as mentioned in this paper.
TL;DR: After binding to cell surface receptors, insulin, along with its receptor, is internalized by an endocytic process, which in contrast to multiplication-stimulating activity is affinity cross-linked to a protein with an entirely different subunit structure.
Abstract: INSULIN receptor has been purified by affinity chromatography and studied by affinity labeling techniques. It is composed of two types of subunits, α (molecular weight ∼135,000) and β (molecular weight α90,000), which form a disulfide-linked heterotetramer (αβ)2. Both α- and β-subunits are glycoproteins. Both are exposed on the outer surface of the membrane, a is the subunit that is predominantly affinity labeled by insulin and is probably the insulin-binding subunit; however, β may also comprise a portion of the insulin-binding site. Receptors for insulin-like growth factors have also been affinity labeled. A photoaffinity labeling derivative of basic somatomedin affinity labels a protein with a subunit structure very similar to the insulin receptor. In contrast, multiplication-stimulating activity is affinity cross-linked to a protein with an entirely different subunit structure. After binding to cell surface receptors, insulin, along with its receptor, is internalized by an endocytic process, which in ...
TL;DR: The proteinase from culture supernatants of Candida albicans strain CBS-2730 was purified virtually to homogeneity by ion-exchange chromatography and affinity chromatography, and only horse ferritin was resistant to proteolysis, while myeloma proteins of the A1- and A2-type were readily cleaved.
TL;DR: The strict requirement of reducing agents for the maintenance of electrolectin agglutination activity is explained by the need to prevent the oxidation of a tryptophan residue in the lactose-binding site.
TL;DR: Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP, which is competitively inhibited by novobiocin and related coumarin antibiotics.
Abstract: Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.
TL;DR: Three lectins have been isolated from an extract of mistletoe by affinity chromatography on partially hydrolysed Sepharose and human immunoglobulin- Separose and all three react with human erythrocytes without specificity for the A, B, and O blood groups.
Abstract: Three lectins have been isolated from an extract of mistletoe (Viscum album) by affinity chromatography on partially hydrolysed Sepharose and human immunoglobulin- Sepharose. The lectins differ in molecular weight and sugar specificity (lectin I, mol.wt. 11500, D-galactose-specific; lectin II, mol.wt. 60000, both D-galactose- and N-acetyl-D-galactosamine-specific; lectin III, mol. wt. 50000, N-acetyl-D-galactosamine-specific). All three lectins react with human erythrocytes without specificity for the A, B, and O blood groups. In contrast with abrin and ricin the mistletoe lectins cannot be divided into "toxins" and "haemagglutinins".
TL;DR: Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.
TL;DR: Human placenta insulin receptor was purified 11,000-fold to near homogeneity using DEAE-cellulose chromatography and affinity chromatography on insulin-Sepharose using disuccinimidyl suberate, suggesting that this component contains the insulin binding domain.
TL;DR: The basic-SM receptor of human placenta is a glycoprotein, remarkably similar to (an isoreceptor) but distinct from the insulin receptor previously characterized in this tissue.
Abstract: Using a recently isolated human basic somatomedin (basic SM) similar to insulin-like growth factor I (IGF-I), we studied both the photoaffinity-labeled and unlabeled basic-SM receptor solubilized from human placental cell membranes. Unlike the result with the insulin receptor, high yields of soluble basic-SM-binding activity are obtained with Triton X-100. The soluble basic-SM receptor retains high-affinity (Kd approximately 0.3 nM) peptide-specific binding of basic SM, similar to the binding present in particulate placenta membranes; the receptor exhibits a comparatively low affinity for insulin (Kd approximately 3 microM). On Sepharose 6B, like the crude soluble insulin receptor, the basic-SM receptor migrates as a species with an apparent Stokes radius of 7.2 nm; unlike the insulin receptor, the basic-SM receptor does not, under similar conditions, yield a smaller binding species (apparent Stokes radius 3.8 nm). Upon photoaffinity labeling with 125I-labeled basic SM, one principal specifically labeled constituent is detected. Upon gel electrophoresis in the presence of 2-mercaptoethanol, the photolabeled constituent, like the insulin receptor, migrates as a species with an apparent molecular weight of about 140,000; in the absence of reducing agent, a molecular weight greater than 240,000 is observed. Lectin-agarose affinity chromatography yields a 30-fold purification both of the basic-SM-binding activity and the photolabeled constituent. Anti-insulin receptor antibody does not appear to precipitate the basic-SM receptor. We conclude that the basic-SM receptor of human placenta is a glycoprotein, remarkably similar to (an isoreceptor) but distinct from the insulin receptor previously characterized in this tissue.
TL;DR: It was demonstrated that NADH, molecular oxygen, linoleoyl-CoA, lipid or detergent, and three enzymes, NADH-cytochrome b 5 reductase, and the terminal enzyme, were absolutely essential for Δ 6 -desaturase, which was found to be a non-heme iron protein containing one atom of iron per one molecule of the enzyme.
TL;DR: 125I-labelled mannan binding to the binding protein is calcium-dependent, and neoglycoproteins containing mannose, N-acetylglucosamine, or L-fucose, but not galactose, are inhibitory.
Abstract: A rat liver mannan-binding protein was isolated by affinity chromatography on invertase--Sepharose by a modification of the method of Kawasaki, Etoh & Yamashina [(1978) Biochem. Biophys. Res. Commun. 81, 1018-1024] and by a new method involving chromatography on mannose-Sepharose. The binding protein appears as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent mol.wt. of approx. 30000. Binding of 125I-labelled mannan is saturable and inhibited by mannose, N-acetylglucosamine, or L-fucose but not by galactose or mannose 6-phosphate. Neoglycoproteins containing mannose, N-acetylglucosamine, or L-fucose, but not galactose, are inhibitory. The neoglycoproteins are 10000-fold more effective (based on moles of sugar) than are free monosaccharides as inhibitors. 125I-labelled mannan binding to the binding protein is calcium-dependent.
TL;DR: Using affinity chromatography and enzyme‐labelled immunological assays combined with aftinity adsorption, evidence is obtained for the binding of a brain glycoprotein to hyaluronic acid, and on this basis it is named hyaluronectin.
Abstract: Using affinity chromatography and enzyme-labelled immunological assays combined with aftinity adsorption, we have obtained evidence for the binding of a brain glycoprotein to hyaluronic acid, and on this basis named it hyaluronectin. This binding was inhibited by hyaluronic acid and by the products of its hydrolysis by hyaluronidase from bovine testis, but was not inhibited by other glycosaminoglycans or by monosaccharides. Preparative affinity chromatography of brain acid-soluble proteins produced hyaluronectin in a good degree of purity. Contamination by albumin was less than 1% and the yield was as high as 80%.
TL;DR: Results indicate that different immobilized naphthalenesulfonamide derivatives interact differently with S100 and calmodulin in a calcium-dependent manner and provide a rapid purification procedure for S-100 andCalmodulin.
TL;DR: Results suggest that only the type II cell synthesizes surfactant protein and than mainly alveolar macrophages participate in its catabolism, and the initial intracellular site of the association of protein with lipid may be multivesicular bodies.
Abstract: We investigated the cellular and subcellular sites of metabolism of the 72,000 dalton protein of pulmonary surfactant in order to provide insights into mechanisms of synthesis, intracellular assembly, and intraalveolar metabolism of this phospholipid-rich secretory product. Surfactant (approximately 90% lipid, 10% protein by weight) was purified by density gradient centrifugation of material obtained by lavaging rat lungs. The purified material was used to generate an antiserum from which a specific antibody was obtained by affinity chromatography. A horseradish peroxidase-labeled Fab was used to localize the antigen in rat lung. The antibody labeled the rough endoplasmic reticulum and Golgi apparatus of type II cells only. Some multivesicular bodies in type II cells were also labeled, but whether the antigen was present in lamellar bodies was uncertain. Phagosomes of alveolar macrophages were labeled as were similar inclusions in type I cells. Using indirect immunocytochemistry we determined that the lab...
TL;DR: When used in conjunction with conventional procedures, affinity chromatography enabled the rapid and specific purification of alpha-galactosidase A from each source, and pyrogenic endotoxins were eliminated from enzyme preparations by the use of the affinity column.
TL;DR: This chapter describes procedure for large-scale purification of cathepsin D, the step include: extraction and homogenization; extraction of acid supernate; chromatography on DEAE-Sephadex A-25 column; affinity column chromatography; and subjection of mixture eluated in the last step to liquid-phase isoelectric focusing.
Abstract: Publisher Summary Cathepsin D is a lysosomal carboxyl (acid) protease widely distributed in many cell types. The presence of this enzyme in relatively high amounts in mammalian spleens has long been recognized. Several purification methods were devised that resulted in pure spleen cathepsin D and in the investigation of its properties.One of the advantages of studying spleen cathepsin D from pigs and cows is that large amounts of starting materials can be obtained; therefore, large-scale purifications can be carried out for the studies of the structure-function relationships of this enzyme. This chapter describes procedure for large-scale purification of cathepsin D, the step include: extraction and homogenization; extraction of acid supernate; chromatography on DEAE-Sephadex A-25 column; affinity column chromatography; and subjection of mixture eluated in the last step to liquid-phase isoelectric focusing. Two methods are used for the assay cathepsin D. The assay using hemoglobin as a substrate is essentially. This assay is rapid and has a reasonable sensitivity (-3000 ng of enzyme). The second assay utilizes renin substrate, and angiotensin I radioimmunoassay. This method, which can readily measure 30 g of cathepsin D, is about 100 times more sensitive than hemoglobin assay, but it requires an elaborate procedure and equipment.
TL;DR: The apparent ubiquitous distribution of mannan-binding protein in mammalian liver is consistent with the proposal that the binding protein is the cellular receptor mediating the hepatic uptake of glycoproteins terminated with mannose and/or N-acetylglucosamine residues.
TL;DR: This chapter describes the preparation of a glutathione-affinity chromatography column that has been successfully used for the purification of the glutathiones-transferases from the human liver.
Abstract: Publisher Summary The glutathione S-transferases from a variety of sources have been purified by the use of conventional chromatographic procedures, as well as by affinity chromatography. When affinity chromatography is used, it is generally necessary to apply additional separation procedures because most of the glutathione S -transferases contain several similar forms of the enzyme. This chapter describes the preparation of a glutathione-affinity chromatography column that has been successfully used for the purification of the glutathiones-transferases from the human liver. This procedure can serve as the initial step in the isolation of the family of glutathione S -transferases from a crude mixture, or it may be used as a final purification step after the several forms of the glutathione S -transferases are separated from one another.