Showing papers on "Affinity chromatography published in 1984"
TL;DR: A tumor-derived growth factor that stimulates the proliferation of capillary endothelial cells has a very strong affinity for heparin, which makes it possible to purify the growth factor to a single-band preparation in a rapid two-step procedure.
Abstract: A tumor-derived growth factor that stimulates the proliferation of capillary endothelial cells has a very strong affinity for heparin. This heparin affinity makes it possible to purify the growth factor to a single-band preparation in a rapid two-step procedure. The purified growth factor is a cationic polypeptide, has a molecular weight of about 18,000, and stimulates capillary endothelial cell proliferation at a concentration of about 1 nanogram per milliliter.
920 citations
Journal Article•
TL;DR: Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG, and this novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.
Abstract: Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.
773 citations
TL;DR: A new natural anti-alpha-galactosyl IgG antibody (anti-Gal) was found to be present in high titer in the serum of every normal individual studied, suggesting a physiological role for this natural antibody in the aging of RBC.
Abstract: A new natural anti-alpha-galactosyl IgG antibody (anti-Gal) was found to be present in high titer in the serum of every normal individual studied. The antibody was isolated by affinity chromatography on a melibiose-Sepharose column. The reactivity of the antibody was assessed by its interaction with alpha-galactosyl residues on rabbit erythrocytes (RabRBC). The specificity was determined by inhibition experiments with various carbohydrates. The anti-Gal interacts with alpha-galactosyl residues, possibly on glycolipids of human RBC (HuRBC), after removal of membrane proteins by treatment with pronase. In addition, the anti-Gal bind specifically to normal and pathologically senescent HuRBC, suggesting a physiological role for this natural antibody in the aging of RBC. The ubiquitous presence of anti-Gal in high titers throughout life implies a constant antigenic stimulation. In addition to the theoretical interest in the antibody, the study of the anti-Gal reactivity seems to bear immunodiagnostic significance. Decrease in the antibody titer was found to reflect humoral immunodeficiency disorders.
682 citations
TL;DR: Brain and pituitary fibroblast growth factors have been purified to apparent homogeneity from crude tissue extracts by a three-step procedure, including salt precipitation, ion-exchange chromatography, and heparin-Sepharose affinity chromatography.
Abstract: Brain and pituitary fibroblast growth factors (FGF) have been purified to apparent homogeneity from crude tissue extracts by a three-step procedure, including salt precipitation, ion-exchange chromatography, and heparin-Sepharose affinity chromatography. Brain and pituitary FGF have similar amino acid compositions and are indistinguishable with respect to molecular weight (16,000 by polyacrylamide gel electrophoresis), retention behavior in reversed-phase high-performance liquid chromatography, and recognition by antibodies directed against the amino-terminal sequence of pituitary FGF. Brain FGF preparations purified by heparin-Sepharose contain, in addition to the major FGF molecular species, at least two additional forms of the growth factor, which appear to be very similar by all the above criteria, except for retention in high-performance liquid chromatography.
597 citations
TL;DR: The human interleukin-2 (IL-2) receptor was purified by affinity chromatography using the anti-Tac monoclonal antibody, and its N-terminal amino acid sequence was determined.
Abstract: The human interleukin-2 (IL-2) receptor was purified by affinity chromatography using the anti-Tac monoclonal antibody, and its N-terminal amino acid sequence was determined. Complementary DNA clones were isolated and sequenced to reveal the primary structure of the IL-2 receptor precursor, which has 272 amino acid residues. The receptor is separated into two domains by a putative 19-residue transmembrane region. Two mRNAs (1.4 and 3.5 kilobases) hybridizing to the cDNA clone were found in human T cells bearing the IL-2 receptor. The cDNA directed synthesis of the IL-2 receptor in COS cells.
551 citations
TL;DR: A monoclonal antibody, named S12, is developed, which demonstrates dramatically enhanced binding to platelets after thrombin activation and suggests that S12 may serve as a useful probe of in vivo platelet activation.
473 citations
TL;DR: Results indicate that platelet secretion is associated with the expression of an Mr = 140,000 integral membrane protein composed of a single polypeptide chain which may be component of the internal granule membrane which is fused with the plasma membrane during activation.
440 citations
TL;DR: A novel myelin antigen has been defined by a mouse monoclonal antibody (8-18C5) raised against rat cerebellar glycoproteins by immunohistochemistry and western blotting followed by immunostaining revealed that the antigen was a glycoprotein of Mr 51 000 daltons which was degraded on storage to cross-reacting products of 20-26 000 dALTons.
316 citations
TL;DR: Results confirm the existence of two distinct growth factors in bovine neural tissue and establish that the acidic endothelial cell growth factor from hypothalamus and the acidic brain fibroblast growth factor are identical.
Abstract: Two growth factors have been purified to homogeneity from either bovine hypothalamus or brain by heparin affinity chromatography. Both stimulate the growth of murine 3T3 fibroblasts and bovine capillary endothelial cells. One heparin-binding growth factor (HGF alpha), purified from either tissue by elution from heparin with 1.0 M sodium chloride, is obtained in a yield of 0.4 mg/kg of tissue. Its apparent molecular weight is 16 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid composition is identical with that of the acidic fibroblast growth factor recently isolated from bovine brain by a multistep chromatographic procedure [Thomas, K. A., Rios-Candelore, M., & Fitzpatrick M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 357-361]. A second growth factor (HGF beta), isolated from either tissue by elution from heparin with 2.0 M sodium chloride, is obtained in a yield of 0.02 mg/kg of tissue. Its apparent molecular weight is 18 000 by SDS-PAGE, and its amino acid composition differs from that of HGF alpha. These results confirm the existence of two distinct growth factors in bovine neural tissue and establish that the acidic endothelial cell growth factor from hypothalamus and the acidic brain fibroblast growth factor are identical.
295 citations
TL;DR: I-Ad, purified from A20-1.11 cells by affinity chromatography, was incorporated into supported planar membranes by incubation of I-Ad-containing phospholipid vesicles with clean glass coverslips and inhibited by the monoclonal antibody MKD6 but not by the antibody 10-2.16 (anti-I-Ak).
Abstract: I-Ad, purified from A20-1.11 cells by affinity chromatography, was incorporated into supported planar membranes by incubation of I-Ad-containing phospholipid vesicles with clean glass coverslips. Such planar membranes present a peptide digest of ovalbumin to the ovalbumin-specific, I-Ad-restricted T-cell hybridoma 3DO-54.8, resulting in the antigen-specific release of interleukin 2. However, when the same material was provided in the form of small unilamellar vesicles, no response was obtained. Antigen presentation by the I-Ad-containing planar membranes was inhibited by the monoclonal antibody MKD6 (anti-I-Ad) but not by the antibody 10-2.16 (anti-I-Ak). The antibody GK1.5, which recognizes the T-cell surface antigen L3T4, was also inhibitory. In contrast to the results with purified I-Ad, crude membrane preparations from A20-1.11 cells were effective in antigen presentation in both planar and vesicular forms.
267 citations
TL;DR: A one-step purification method of hybrid proteins exhibiting β-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described, which can be used to obtain antibodies against the foreign portion of the protein fusion.
TL;DR: Its synthesis by normal mesenchymal cells and by malignant or transformed cells of both ectodermal and endodermal origin suggests a general role in cell function that is independent of transformation.
TL;DR: Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods and specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized.
Abstract: Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods. Complexes of RII and its binding proteins were isolated on a cAMP analog-Sepharose affinity column, eluted from the column, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Alternatively, nitrocellulose blots made from polyacrylamide gels containing samples of tissue extracts or affinity column eluates were treated with sequential overlays of RII, monospecific antibody, and radioiodinated protein A. In bovine cerebrum, specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized by the two methods. The 300-kDa and 68-kDa proteins were identified as microtubule-associated protein 2 (300 kDa) and a protein of lower molecular weight (68 kDa) that copurifies with it. The additional major binding protein of 80 kDa requires further characterization. In addition, several binding proteins distinct from those observed in bovine cerebrum were found in bovine heart. Many of the RII binding proteins from brain and heart served to differing extents as substrates for the purified catalytic subunit of cAMP-dependent protein kinase. One hypothesis of the significance of the protein kinase regulatory subunit interaction with cellular binding proteins is that this may control the protein kinase holoenzyme localization and, thereby, define the substrate targets most accessible for phosphorylation by the activated protein kinase catalytic subunit. Alternatively, RII binding to a variety of cellular proteins might regulate their function--i.e., RII could be a regulator for multiple proteins in addition to the catalytic subunit of the cAMP-dependent protein kinase.
TL;DR: The placenta radioreceptorassay for somatomedin can be used for detection of the binding protein, which has a high content of acidic/amidated residues and was isolated from human amniotic fluid by a three-step procedure.
Abstract: Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl-Sepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%. Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.
TL;DR: Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where the effect of varying the concentration of thrommodulin with respect to throm bin and the converse, on rates of protein C activation.
TL;DR: Three unique fragments of 83,000, 42,000 and 19,000 mol wt are present and they represent the major surface antigens of P. falciparum merozoites and are used to identify the terminal processing products associated with the merozosite.
Abstract: A 195,000 mol wt Plasmodium falciparum protein and processing fragments derived from it have been purified by monoclonal antibody affinity chromatography. A polyvalent antiserum has been raised against the purified protein and used to identify the terminal processing products associated with the merozoite. Three unique fragments of 83,000, 42,000, and 19,000 mol wt are present and they represent the major surface antigens of P. falciparum merozoites.
TL;DR: Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.
Abstract: The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.
TL;DR: Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid or Ca2+ was performed, allowing a definitive study of the ability of these fragments to activate, or interact with, cal modulin-regulated enzymes and anti-calmodulin drugs.
TL;DR: The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells and were readily partially purified by immunoaffinity chromatography from lysates of infected Cells solubilized with denaturing agents such as sodium dodecyl sulfate.
Abstract: Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes simplex virus (HSV) 1 and 2 and by HSV-1 X HSV-2 recombinants indicated that monoclonal antibody H1183 reacted with HSV-1 alpha ICP0, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 alpha ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described (D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these alpha proteins. The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICP0 was considerably more soluble than ICP4 and ICP27. ICP0, ICP4, and ICP27 were readily partially purified by immunoaffinity chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl sulfate. ICP0 and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h) or late (18 to 20 h) postinfection. A fraction, but not all, 32P that was incorporated early was chased in the presence of unlabeled phosphate. ICP0, ICP4, and ICP27 labeled with either 32P or [35S]methionine yielded multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at the origin when the direction of migration was reversed.
TL;DR: Data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2-plus-phorbol ester-dependent interaction.
TL;DR: Both purified forms of the enzyme possessed substantial glutathione S -transferase activity with both CDNB and DCNB, and the same protein generated a number of minor protein bands on analytical electrofocusing in polyacrylamide gels, but there is evidence that these bands may be artifactual.
TL;DR: Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups, which is about 10-fold lower than that for phosphorylation of exogenous substrates.
TL;DR: Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Separose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparIn-binding proteins.
TL;DR: Developpement d'une methode qui combine les avantages de la chromatographie d'affinite et de the chromatographs en phase liquide de haute performance: specificite, rapidite et sensibilite.
TL;DR: Data in this and the accompanying paper provide strong evidence that erythrocyte acetylcholinesterase is an amphipathic protein.
TL;DR: A trypsin-like, membrane-bound protease from Bacteroides gingivalis was solubilized by Triton X-100 and partially purified by a combination of DEAE-Sepharose and aminophenylmercuric Sepharose chromatography, by taking advantage of the thiol group on the enzyme.
TL;DR: This chapter discusses an immunological approach to detect tyrosine-sulfated proteins and on the determination of tyosine sulfate by methods that are based on labeling proteins with 35 SO 4.
Abstract: Publisher Summary This chapter focuses on the detection of tyrosine-sulfated proteins and on the determination of tyrosine sulfate by methods that are based on labeling proteins with 35SO4. This approach has several advantages over the chemical determination of tyrosine sulfate in unlabeled proteins. It can be much more easily used to search for tyrosine-sulfated proteins and is considerably more sensitive, and therefore requires less protein material for analysis. The chapter discusses an immunological approach to detect tyrosine-sulfated proteins. In this approach, antisera were raised against a synthetic antigen containing a large number of tyrosine sulfate residues and antibodies were purified from the antisera by affinity chromatography. These antibodies appeared to recognize tyrosine sulfate-containing proteins in “Western” blots and in solid-phase radioimmunoassay. The general usefulness of these antibodies to screen for, immunoprecipitate, or purify tyrosine-sulfated proteins is currently being tested. The standard method to detect tyrosine sulfate in proteins involves four main steps: (1) in vivo labeling of tissues in situ or of tissue explants, tissue slices, and cells in culture with inorganic [35S]sulfate; (2) the separation of proteins by polyacrylamide gel electrophoresis (PAGE); (3) elution from gels and the hydrolysis of individual 35SO4-1abeled proteins; and (4) the separation of tyrosine [35S]sulfate by thin-layer electrophoresis.
TL;DR: Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH.
Abstract: Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.