Showing papers on "Affinity chromatography published in 1982"
TL;DR: It is apparent that phenyl-SepHarose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin, and the time required for this procedure is substantially less than that of previously described procedures.
840 citations
TL;DR: Results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently, in epidermal growth factor receptor-kinase complex from A-431 cells.
770 citations
TL;DR: The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione, and this protein appears to be distinct from the selenoenzyme glutATHioneperoxidase and from any known glutathion S-transferase.
668 citations
TL;DR: The ability of E-PHA and L- PHA to discriminate between Asn-linked oligosaccharides with various branching patterns can be utilized in the fractionation of these glycopeptides (see paper following).
524 citations
TL;DR: It is concluded that serial lectin-agarose affinity chromatography is a rapid, sensitive, and specific technique for fractionating and analyzing Asn-linked oligosaccharides.
485 citations
TL;DR: The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose and an overall purification of 1950-fold was achieved.
368 citations
TL;DR: A rapid two-step procedure for the purification of the low density lipoprotein receptor from bovine adrenal cortex membranes yields a single protein with a molecular weight of 164,000, which retains all of the binding properties of the receptor of intact cells and crude membranes.
354 citations
TL;DR: It is concluded that the HEp 3 protein is a proenzyme that can be converted to active two-chain urokinase by plasmin, probably by a single proteolytic nick in the polypeptide chain.
335 citations
TL;DR: Data indicate that a covalent, possibly thiolester intermediate, is formed between the activating enzyme and Sepharose-bound ubiquitin, and it is suggested designating this procedure of enzyme isolation "covalent affinity" chromatography.
315 citations
TL;DR: Simplified procedures have been developed for isolation of human plasma fibronectin by affinity chromatography on gelatin-agarose, and the fraction not adsorbed to gelatin can be used to purify other proteins, including factor VIII whose procoagulant activity is quantitatively recovered.
313 citations
TL;DR: There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquin than most normal proteins.
TL;DR: The purification of p97 by affinity chromatography with monoclonal antibody, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and determination of the N-terminal amino acid sequence using a new, highly sensitive protein sequencer found the sequence to be homologous to the N -terminal sequences of transferrin and lactotransferrin.
Abstract: p97 is a 97,000-molecular weight (MW) cell-surface glyco-protein, which is present in most human melanomas but in only trace amounts in normal tissues1–4. We describe here the purification of p97 by affinity chromatography with monoclonal antibody, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and determination of the N-terminal amino acid sequence using a new, highly sensitive protein sequencer5. The sequence was found to be homologous to the N-terminal sequences of transferrin and lactotransferrin. This structural homology was confirmed by the observation that antiserum to denatured p97 cross-reacted with denatured transferrin and lactotransferrin. We have also demonstrated that p97 is functionally related to transferrin and lactotransferrin in that it binds iron. This is one of the first reports of the amino acid sequence of a human tumour-associated cell-surface antigen and one of the few cases in which insight has been obtained into its function.
TL;DR: Findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain.
Abstract: Incorporation of the serine protease active site reagent diisopropyl fluorophosphate (DFP) into a plasminogen activator with an Mr of approximately 52000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (NaDodSO4), having one stainable band under nonreducing as well as reducing conditions with an Mr of approximately 52000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After NaDodSO4-polyacrylamide gel electrophoresis, the active enzyme showed one band under nonreducing conditions, but after reduction, two bands with Mr values of approximately 20000 and 32000 were observed. The active enzyme incorporated [3H]DFP into the approximately Mr 32000 band, while no incorporation was observed into the inactive form. These findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher molecular weight form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence.
TL;DR: Immunofluorescence studies and cell fractionation of MC29-transformed fibroblasts indicate that the 110 K protein is predominantly located in the nucleus, and the purified protein binds to double-stranded DNA, which may be related to the role of the protein in transformation.
Abstract: Oncornaviruses transform cells either directly through a virus-coded oncogene1 or, if they lack such a gene, indirectly by promoter insertion2–4 into the cellular genome and activation of a cellular gene. Both transformation mechanisms ultimately result in abnormally high expression of normal genes. Recently, the activated cellular gene in certain lymphomas4 has been shown to be homologous to the oncogene of an acute avian leukaemia virus, MC29. In MC29 the oncogene is fused to the viral structural gene, gag. The product of this fused gene is a protein of molecular weight 110,000 (110 K)5–7, designated p110gag–myc. We have characterized this protein by using monoclonal antibodies against p19, the N-terminal portion of the gag–myc fusion or 110 K protein and purified it 3,700-fold by immune affinity column chromatography. Immunofluorescence studies and cell fractionation of MC29-transformed fibroblasts indicate that the 110 K protein is predominantly located in the nucleus. Moreover, the purified protein binds to double-stranded DNA. These properties may be related to the role of the protein in transformation.
TL;DR: The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many commonerences.
Abstract: An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.
TL;DR: Avian luteinizing hormone-releasing hormone (LH-RH) has been isolated from 249,000 chicken hypothalami and shown to differ structurally from mammalian hypothalamic LH-RH, and synthesized and established that it has chromatographic properties identical with natural chicken LH- RH.
TL;DR: This chapter presents procedure for purification and characterization of PAI and PAII, which involves crude extraction from bacteria grown in Grelet's medium, ammonium sulfate precipitation, and chromatography on Sepharose 4B.
Abstract: Publisher Summary Extracts of certain strains of Pseudomonas aeruginosa contain two lectins: PAI and PAII. This chapter presents procedure for purification and characterization of PAI and PAII. The PAI purification involves crude extraction from bacteria grown in Grelet's medium, ammonium sulfate precipitation, and chromatography on Sepharose 4B. The yield of the purified PA-I is relatively high and is practically free of PA-II. PA-II is purified from the extracts of the bacteria grown in nutrient broth. The first two steps of its purification are the same as for PA-I: heating to 70 ° and precipitation by ammonium sulfate, as described above. The dissolved ammonium sulfate precipitate is purified by affinity chromatography on D-mannose-bearing Sepharose 4B. Thirty milliliters of the lectin preparation are loaded onto a column (4 x 20 cm) containing the modified Sepharose. The activity of the lectins is determined by measuring their ability to agglutinate neuraminidase or papain-treated human erythrocytes. The hemagglutination assay can also be used as an indirect demonstration of lectin interaction with nonagglutinable cells by determining the decrease in the hemagglutinating activity of the lectin preparations as a result of their exposure to the examined cells. The interaction of PA-II with cells can also be detected by a peroxidase-binding assay.
TL;DR: These monoclonal antibody preparations, recognizing different determinants on the estrophilin molecule, provide a novel approach to the study of receptor structure and function as well as the basis for a simple immunoradiometric determination of estrogen receptors in human breast cancers.
TL;DR: The lectin from the seeds of Erythrina cristagalli has been isolated in high yield and homogeneous form by affinity chromatography on a column of D-galactose-derivatised Sepharose, and the difference spectrum obtained upon binding of the disaccharide shows maxima at 291 nni and 282-284 nm, indicating a change in the environment of tryptophan residues of the protein uponbinding of sugar.
Abstract: The lectin from the seeds of Erythrina cristagalli has been isolated in high yield (75 %) and homogeneous form by affinity chromatography on a column of D-galactose-derivatised Sepharose. It is a glycoprotein with a molecular weight of 56800±900 and s20.w, = 3.9 S, composed of two subunits (apparent molecular weights of 28000 and 26000 respectively) both of which are glycosylated. The total carbohydrate content is 4.5% and it is comprised of mannose, N-acetylglucosamine, fucose and xylose in amounts corresponding to 7, 4, 2 and 2 mo1/56800 Da respectively. The amino acid composition of the lectin is characterised by a high content of acidic and hydroxy amino acids, low content of methionine and absence of cysteine. Valine is the only N-terminal amino acid detected. The lectin is a metalloprotein in that it contains 0.093% Mn and 0.13percnt; Ca (1 mol and 1.9 mo1/56 800 Da respectively), both of which are tightly bound to the protein.
E. cristagalli lectin agglutinates untreated human erythrocytes of all blood types, as well as rabbit erythrocytes, at a concentration of 5-10 μg/ml. It is mitogenic for human peripheral blood T lymphocytes at an optimal concentration of about 100 pμg/ml, but is not mitogenic for mouse thymocytes or splenocytes.
d-GahtOSe and various d-galactosides inhibit the hemagglutinating activity of the lectin. N-Acetyllactosarnine is most potent, completely inhibiting four agglutinating units of the lectin at 0.4 mM concentration. Lactose, N-acetyl-d-galactosamine and d-galactose are 5, 16 and 35 times less active respectively. Lactose specifically perturbs the ultraviolet spectrum of the lectin in the aromatic region. The difference spectrum obtained upon binding of the disaccharide to the lectin shows maxima at 291 nni and 282-284 nm, indicating a change in the environment of tryptophan residues of the protein upon binding of sugar.
TL;DR: Bovine submaxillary mucin, a sialoprotein, was a potent inhibitor of hemagglutination by LFA and loss of inhibitory activity was observed which was proportional to the loss of sialic acid from the mucin.
TL;DR: The usefulness of the cytoplasmic inhibitor of RNase to protect RNA in the course of the synthesis of complementary DNA by reverse transcriptase is discussed.
Abstract: Publisher Summary Bovine Ribonuclease has been a test protein in the study of a wide variety of chemical and physical methods of protein chemistry. It is widely employed in the course of the sequencing of RNA. This chapter discusses the usefulness of the cytoplasmic inhibitor of RNase to protect RNA in the course of the synthesis of complementary DNA by reverse transcriptase. It examines the use of the inhibitor to protect mRNA during the course of in vitro translation and during the preparation of rough microsomes and detached polysomes. The introduction of affinity chromatography has simplified the isolation of pure RNases by providing a highly efficient method for separating active enzymes from molecules that do not have an affinity for the coupled substrate analog. The technique can be used as an early step in purification or as a final step after ion exchange chromatography and gel filtration to isolate an RNase fraction of given charge and size.
TL;DR: The carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs appears to be a new isozyme whose properties are consistent with such a localization.
TL;DR: A Ca2+-dependent protease was prepared from rat brain by using DEAE-Sephadex, SephadeX G-200, and substrate affinity chromatography and degradation of neurofilament proteins was determined by measuring the changes in radioactivity of electrophoretically separated bands of radioiodinated neuro Filament proteins.
Abstract: A Ca2+-dependent protease was prepared from rat brain by using DEAE-Sephadex, Sephadex G-200, and substrate affinity chromatography. Degradation of neurofilament proteins was determined by measuring the changes in radioactivity of electrophoretically separated bands of radioiodinated neurofilament proteins. The apparent Km values for 68000- (P68), 150000- (P150), and 200000- (P200) dalton neurofilament proteins are 3.9 x 10(-8) M, 4.4 x 10(-8) M, and 8.2 x 10(-8) M, respectively. Proteolytic activity is dependent upon Ca2+ concentration with threshold and saturation values of 10(-6) and 10(-4) M, respectively. The enzyme is also inactivated by preincubation with Ca2+. Similar Ca2+ concentrations cause activation and inactivation of enzyme, but the process of inactivation is intrinsically slower than the process of activation. The enzyme is sensitive to thiol protease inhibitors, is activated by Sr2+, Ba2+, Mn2+, and La3+ at 1-10 mM, and has an optimal pH range of 7.4-8.0.
TL;DR: Results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.
Abstract: The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.
TL;DR: Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer, and protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin.
TL;DR: This chapter discusses the purification of rhodopsin by concanavalin a affinity chromatography, a technique based on the reversible binding of a ligand to a protein that represents one of the major advances in protein purification methodology.
Abstract: Publisher Summary This chapter discusses the purification of rhodopsin by concanavalin a affinity chromatography. Affinity chromatography represents one of the major advances in protein purification methodology. This technique depends on the reversible binding of a ligand to a protein. Rhodopsin has been identified as a mannose-containing glycoprotein and as such has the potential ligand properties for affinity chromatographic purification. The procedure to be described employs the nonionic detergent octyl glucoside. Rhodopsin fractions are pooled and can be concentrated by ultrafiltration using a Diaflo PM 10 or PM 30 filter. Pooled samples are generally concentrated to about 3 mg/ml of rhodopsin, involving a reduction in volume from about 40 ml to 5 ml. This process results in a concentration of octyl glucoside and rhodopsin, the final detergent concentration being in the range of 60 mM to 70 mM; this raises no concern with respect to rhodopsin stability, as rhodopsin is stable in octyl glucoside solutions as high as 300 mM.
TL;DR: The chapter presents two purification procedures for maltose-binding protein—growth of cell and osmotic shock treatment, and QAE-Sephadex chromatography, and the alternative purification procedure is affinity chromatography.
Abstract: Publisher Summary This chapter describes the assay method, the purification procedure, and the properties of maltose-binding protein from Escherichia coli . The maltose-binding protein is a periplasmic protein of Escherichia coli that is involved in the transport of maltose and maltodextrins across the bacterial envelope and in the chemotaxis toward these sugars. Maltose binding is measured by equilibrium dialysis using [ 14 C]maltose. The chapter presents two purification procedures for maltose-binding protein—growth of cell and osmotic shock treatment, and QAE-Sephadex chromatography. The alternative purification procedure is affinity chromatography. Both methods yield a protein that is more than 90% pure as estimated by electrophoresis on polyacrylamide gels in different conditions. A molecular weight of 40,000 to 44,000 of the protein is determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The maltose binding protein is coded by gene malE , one of the five cistrons of the malB region involved in the transport of maltose and maltodextrins across the envelope of E.coli.
TL;DR: The hypothesis that there is an intrinsic structural difference between monoamine oxidase A and B is supported and it is demonstrated that immunoaffinity chromatography can physically resolve the two enzyme species in liver extracts.
Abstract: A monoclonal antibody was used to prepare immunoaffinity columns that efficiently bind monoamine oxidase B activity but not monoamine oxidase A activity from detergent extracts of human liver mitochondria. The only discrete polypeptide component that eluted from affinity columns with potassium thiocyanate migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 59,000, as expected for human monoamine oxidase B. These results support the hypothesis that there is an intrinsic structural difference between monoamine oxidase A and B and demonstrate that immunoaffinity chromatography can physically resolve the two enzyme species in liver extracts.
TL;DR: In this paper, the authors described a procedure for the isolation of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP).
TL;DR: A complex between the purified S. aureus protein and fibronectin could be demonstrated by crossed immunoelectrophoresis both in monospecific antibodies against fibronECTin and in S.aureus polyspecific antibody.
Abstract: Fibronectin ("cold-insoluble globulin") has been suggested to play a role in cell-to-cell and cell-to-substratum adhesions. The 70-kilodalton terminal part of human fibronectin has recently been shown to bind to Staphylococcus aureus. In the present study, a fibronectin-binding protein was purified from sonicated S. aureus strain E2371 by affinity chromatography on fibronectin-Sepharose. The fibronectin-binding protein was isolated from an extract of sonicated S. aureus containing at least 57 different proteins as determined by crossed immunoelectrophoresis in antibodies to sonicated S. aureus. The fibronectin-binding protein was released from fibronectin-Sepharose by carbamide (8 M). No impurities in the final preparation could be detected when tested in crossed immunoelectrophoresis. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed two bands with relative molecular masses of 197,000 and 60,000, respectively. A complex between the purified S. aureus protein and fibronectin could be demonstrated by crossed immunoelectrophoresis both in monospecific antibodies against fibronectin and in S. aureus polyspecific antibody.