Showing papers on "Affinity chromatography published in 1978"
TL;DR: Activation of cAMP phosphodiesteriase by the Ca2+-dependent activator protein may be controlled by interactions with yet a third component of the enzyme complex.
Abstract: The Ca2+-dependent, reversible, interaction of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase with its activator has been used to purify the enzyme by affinity chromatography. Activator-dependent cAMP phosphodiesterase is only a minor component of the proteins specifically adsorbed in the presence of Ca2+ by the Ca2+-dependent activator protein coupled to Sepharose and subsequently released by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The major protein component can be partially resolved from the enzyme by gel filtration on Sephadex G-200. This protein has been purified to apparent homogeneity and shown to be composed of two polypeptide chains with molecular weights of 61,000 and 15,000 respectively. This protein is, by itself, devoid of phosphodiesterase activity and inhibits the activation of cAMP phosphodiesterase by its activator without affecting the basal activity. Thus, activation of cAMP phosphodiesteriase by the Ca2+-dependent activator protein may be controlled by interactions with yet a third component of the enzyme complex.
422 citations
TL;DR: The kinetic properties of the enzyme suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions and this prediction was confirmed experimentally.
Abstract: Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP+, but GSH-dependent NADP+ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn2+. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 μM and that for NADPH was about 3 μM. NADP+ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP+-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H2O2. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.
398 citations
TL;DR: It is proposed that the mannan-binding protein is the principle mediating the hepatic uptake of glycoproteins with terminal N-acetylglucosamine and/or mannose residues which are cleared rapidly from the circulation by the liver.
324 citations
TL;DR: The procedure reported in the chapter describes the purification to apparent homogeneity of NADPH cytochrome P-450 reductase by solubilization with Renex 690 and affinity chromatography.
Abstract: Publisher Summary This chapter provides information on the purification and properties of NADPH-cytochrome P-450 reductase. NADPH—cytochrome P-450 reductase, a flavoprotein component of the endoplasmic reticulum of liver and other organs, catalyzes the transfer of electrons from NADPH to cytochrome P-450. Cytochrome P-450 is the terminal oxidase of the drug metabolism system that hydroxylates a variety of compounds, such as alkanes, fatty acids, drugs, and steroids. Several forms of this hemoprotein are purified to homogeneity, differing in minimum molecular weight and substrate specificity. The procedure reported in the chapter describes the purification to apparent homogeneity of NADPH cytochrome P-450 reductase by solubilization with Renex 690 and affinity chromatography. The affinity column used is an NADP ligand attached to Sepharose 4B through adipic acid dihydrazide. The reduction of cytochrome P-450 can be determined directly under anaerobic conditions in the presence of carbon monoxide by following the formation of the peak at 450 nm in the reduced carbon monoxide difference spectrum.
293 citations
TL;DR: In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel‐filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.
Abstract: Serum samples and immunoglobulin fractions of eight mammalian species were applied to a Sepharose-protein A column. As with the human immunoglobulin subclasses IgG1, IgG2 and IgG4, all examined animal IgG classes and subclasses were bound to a greater or lesser extent to protein A. However, the binding of IgG1 of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reactive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.
286 citations
TL;DR: Heteroclitic property as well as the presence of λ, μ and γ1 chains are characteristic for primary anti‐NP sera of C57BL/6 mice.
Abstract: Spleen cells from C57BL/6 mice sensitized to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were hybridized with myeloma cells, and a variety of hybrid cell lines was isolated which secreted homogeneous anti-NP antibodies. The antibodies were purified by affinity chromatography and their chain composition, affinity and fine specificity were determined. All antibodies recovered from the primary immune response carried λ light and μ or γ1 heavy chains. Their variable portions were nonidentical but similar in terms of hapten-binding specificity with a higher affinity for the cross-reacting haptens (4-hydroxy-3,5-dinitro-phenyl)acetyl (NNP) and (4-hydroxy-5-iodo-3-nitro-phenyl)acetyl (NIP) than for the homologous hapten NP. This heteroclitic property as well as the presence of λ, μ and γ1 chains are characteristic for primary anti-NP sera of C57BL/6 mice.
In contrast, four families of anti-NP antibodies, each with a characteristic fine specificity pattern, were found among the hybrid cell antibodies derived from the hyperimmune anti-NP response. The antibodies of one of these families were related to the antibodies recovered from the primary immune response in that they were heteroclitic and carried λ light chains. All members of the other groups expressed k chains and were nonheteroclitic.
The finding of well-defined antibody families in this system and the isolation of their members enable us to approach the problem of V gene expression and diversification in a new way.
285 citations
TL;DR: Mouse interferon has been purified to homogeneity by two-step affinity chromatography by using 8×109 of the authors' laboratory units corresponding to 2.4×109 NIH reference units for antiviral activity.
Abstract: Mouse interferon has been purified to homogeneity by two-step affinity chromatography. Two polypeptide bands were obtained on sodium dodecyl sulphate–polyacrylamide gel electrophoresis migrating at molecular weights 35,000 and 22,000, both having antiviral activity. The 35,000 but not the 22,000 band, also stained with periodic acid–Schiff. The specific activity was 8×109 of our laboratory units, corresponding to 2.4×109 NIH reference units.
200 citations
TL;DR: High Performance Liquid Afinity Chromatography (HPLAC) and its Application to the Separation of Enzymes and Antigens
172 citations
TL;DR: It is shown that a solid-phase immune adsorbent can be prepared from rabbit antiserum by isolating the IgG fraction with the A protein of Staphylococcus aureus associated covalently with a Sepharose matrix using the cross-linking agent dimethylsuberimidate.
144 citations
TL;DR: Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar, and rapid denaturation of the enzyme is suggested.
140 citations
TL;DR: A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%.
Abstract: A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) (‘DT-diaphorase’, EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
TL;DR: It is suggested that this protein is involved in the development of the capacity for motility as sperm traverse the epididymis as well as other studies on the activity, properties, and distribution of forward motility protein in bovine body fluids.
TL;DR: Purified C-reactive protein was shown to be homogeneous by electrophoretic, immunochemical and ultracentrifugal criteria and by sedimentation equilibrium a molecular weight of 118,000 was calculated in neutral buffer.
TL;DR: Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield and appears to consist of three subunits of Mr = 83,000, 55,000; and 49,000 and contains a significant amount of bound zinc.
TL;DR: Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related, but the alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolate from rat liver.
TL;DR: A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-SepHarose 4 B, chromatographyon Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing.
Abstract: A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.
TL;DR: The bound and eluted fraction of the labeled fibronectin showed more than 90% binding to antibody and to collagen, which suggests that the avidity of the antibody-fibronECTin interaction is higher than that between fibronsectin and collagen.
TL;DR: Evidence was obtained that the inhibitor formed a complex with both dithiothreitol-activated papain and enzymatically inactive mercuripapain, and yielded a highly specific anti-inhibitor serum.
TL;DR: An active preparation of lecithin: cholesterol acyltransferase (LCAT) was isolated from human plasma by density ultracentrifugation, high-density lipoprotein affinity chromatography, DEAE-Sepharose and hydroxylapatite chromatography and apolipoprotein D does not appear to be an activator of LCAT.
Abstract: A homogeneous preparation of active lecithin:cholesterol acyltransferase (LCAT EC 23.1.43) was isolated from human plasma by density ultracentrifugation, high density lipoprotein affinity chromatography, DEAE-Sepharose. and hydroxylapatite chromatography. The enzyme with an apparent molecular weight of 66,000 ± 2,000 was characterized by a high content of glutamic acid, aspartic acid, leucine and glycine, and contained approximately 35 moles of glycosamine/105 g protein and no galactosamine. The purified enzyme, stored at 40 µg/ml at 4 °C, had a half life of 18-30 days. The effect of purified human plasma apolipoproteins A-I, A-II, C-I, C-II, C-III and D on the activity of purified LCAT was studied, using egg-yolk lecithin (40 nM):cholesterol (10 µM) vesicles prepared in 1.25% ethanol in the absence or presence of 0.5% albumin. Significant transacylation occurred only with apoA-I or apoC-I, with maximum activation at 0.18-0.44 µM A-I (n = 11), and 0.71 µM C-I (n = 9). Addition of albumin to the incubation...
TL;DR: Results tend to support the dynamic conversion of T3 T1 T2 rather than their existence as separate enzymes.
Abstract: Studies of mammalian tyrosinase have been conducted principally with soluble forms (T1 faster, T2 slower migrating by gel electrophoresis). However, a majority of tyrosinase from mouse and human melanoma tissue is of a particulate (T3) nature, being bound to melanosomes. In addition, T1 and T2 enzymes have been considered isozymes with T3 being a separate enzyme.
Using human malignant melanoma tissue, solubilization and purification of particulate tyrosinase have been attempted. Melanosome-bound tyrosinase was solubilized with sodium cholate and purified 1500-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, concanavalin A affinity chromatography, and Sephadex G-150 gel filtration. Tryptic cleavage of this solubilized tyrosinase yielded a fast migrating tyrosinase by gel electrophoresis. This trypsin-cleaved tyrosinase was purified by Sephadex G-150 and DEAE-cellulose columns with an apparent final yield of 56% and an approximate 10000-fold purification. This preparation gave a single band by 7% gel electrophoresis and 5% sodium dodecyl sulfate gel electrophoresis. In addition, electrophoresis indicated that this tyrosinase was a glycoprotein showing positive staining by periodic acid/Schiff stain. This enzyme oxidized both l-tyrosine and l-3,4-dihydroxyphenylalanine at a similar rate. The molecular weight was approximated at 66 700 by sodium dodecyl sulfate gel electrophoresis. This enzyme migrated to the T1 position by gel electrophoresis; however, upon treatment with neuraminidase, it shifted to the T2 position. Sialic acid content was determined as 4.8%. Copper content was estimated as 2 atoms/molecule. These results tend to support the dynamic conversion of T3 T1 T2 rather than their existence as separate enzymes.
TL;DR: The results support the hypothesis that H2Se or a similarly reduced selenide is the product of selenite metabolism by rat erythrocytes, and the properties of the Cd-Se complex suggested that it existed in a single form associated with different plasma components under various conditions.
TL;DR: A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.24) from calf brain has been developed using affinity chromatography as mentioned in this paper.
TL;DR: By this method the best purification of human prostatic acid phosphatase so far is achieved, and the enzyme had been isolated in the dimer form.
Abstract: The main isoenzyme of human prostatic acid phosphatases was purified by affinity chromatography on L(+)-tartrate linked to agarose and by isoelectric focusing. The enzyme was a single protein when examined by polyacrylamide gel electrophoreses, either as a native protein or in the presence of sodium dodecyl sulfate. The analytical recovery of enzyme activity was 19%. The specific activity was 40 18 mumol/(min X mg) for hydrolysis of 5.5 mmol/liter p-nitrophenyl phosphate at pH 4.8 and 37 degrees C. The purification factor was as great as 1900. The molecular weight of the enzyme as measured by gel filtration was 109 000 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate 54 000, indicating that the enzyme had been isolated in the dimer form. By this method we have achieved the best purification of human prostatic acid phosphatase so far.
TL;DR: The cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein; this suggests that synthesis of both RNA and protein are impaired.
Abstract: Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6-thioguanosine or 4-thiouridine followed by separation of thiol-containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6-thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)-containing and poly(A)-free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4-thiouridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6-thioguanosine are less stable than proteins synthesised after treatment of cells with 4-thiouridine.
TL;DR: Following chromatography on protein A-Sepharose, immunoglobulin G preparations were devoid of contaminating serum proteins, in particular ribonuclease activity, that are not normally removed using conventional techniques of salt precipitation in combination with ion-exchange chromatography.
TL;DR: It is concluded that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.
Abstract: In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.
TL;DR: Experiments on the coupling of glycidyl methacrylate gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group, and the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent.
TL;DR: Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.
Abstract: Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine. Agarose coupled to L-histidyldiazobenzylphosphonic acid was found to be a highly effective adsorbent. In order to understand the large differences in binding capacity observed with derivatized agaroses, inhibition of alkaline phosphatase by phosphonic acid ligands, and related phosphonic acids, was measured. The results of affinity chromatography and inhibition studies were in good agreement, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.
TL;DR: Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers.
TL;DR: The lectin from Datura stramonium can be inhibited by oligomers of N-acetylglucosamine and this property was exploited to purify the lectin by affinity chromatography on Sepharosefetuin.
Abstract: The lectin from Datura stramonium can be inhibited by oligomers of N-acetylglucosamine. This property was exploited to purify the lectin by affinity chromatography on Sepharosefetuin. The purified lectin is a glycoprotein in having subunits of 40 000 and 45 000 mol.wt.