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At least 19 recordsLinked to original sources
PubMed1984

Plasma therapy of primary rat mammary carcinoma: antitumor activity of tumor-bearer plasma adsorbed against inactivated CNBr sepharose or protein A-sepharose.

We evaluated the ability of plasma adsorbed against Sepharose, inactivated CNBr Sepharose, or protein A-Sepharose to inhibit the growth of primary mammary carcinomas in Buffalo/N rats. Mammary adenocarcinomas were induced by a single intravenous injection of N-nitroso N-methylurea. When palpable mammary tumors were detected, rats were treated by infusion of plasma obtained from normal or tumor-bearing rats. Animals were killed 50 days after entry into the experiment; the index mammary tumor and any subsequently arising tumors were weighed and examined histologically. Unadsorbed plasma obtained from normal or tumor-bearing rats had no detectable antitumor effect. Normal plasma adsorbed against Sepharose or Sepharose derivatives had no significant antitumor effect. Weight (median) of index mammary tumors in groups of rats treated with normal plasma adsorbed against Sepharose alone, inactivated CNBr Sepharose alone, or protein A--Sepharose alone was not significantly different from weight of tumors of untreated, control animals. Tumor-bearer plasma adsorbed against either inactivated CNBr Sepharose or protein A-Sepharose had significant antitumor effect. Weight of index mammary tumors in groups of rats treated with tumor-bearer plasma adsorbed to inactivated CNBr Sepharose alone or against protein A-Sepharose alone was significantly less than weight of tumors in control rats. Tumor-bearer plasma adsorbed against Sepharose alone inhibited tumor growth in one experiment but not in a second experiment. Tests for endotoxin in CNBr Sepharose indicated the presence of approximately 1 ng endotoxin/g Sepharose. Administration of nanogram quantities of Salmonella enteritidis endotoxin in saline did not inhibit growth of primary mammary tumors. These experiments describe a rodent model that may be useful in the analysis of the basis of the inhibition of tumor growth that occurs following administration of adsorbed plasma.

Animals↗
PubMed2005

Interactions of alcohol dehydrogenase to p-hydroxyacetophenone-sepharose and p-acetamidophenol-sepharose.

BACKGROUND: p-Hydroxyacetophenone (HAP)-sepharose is known to be an effective ligand for isolation of aldehyde dehydrogenase and chloramphenicol acetyltransferase. In this study, we investigated ligand specificities to alcohol dehydrogenase (ADH) using p-HAP-sepharose and p-acetamidophenol (AAP)-sepharose. METHODS: p-HAP and p-AAP were coupled to epoxy-activated sepharose and used as ligands for affinity chromatography to detect binding proteins. Fractions of affinity chromatography were collected and separated by SDS-PAGE. Commassie brilliant blue staining was carried out for detecting proteins. For determining protein sequences, polypeptide was separated by HPLC after cleavage of Lys-specific protease digestion. RESULTS: p-HAP ligands have the ability to bind to seven proteins, including class I ADH extracted from a rat cytosolic fraction as well as HLADH (horse liver ADH). p-HAP, p-AAP and benzaldehyde (10 mM each) could elute HLADH from the complex of HLADH-pHAP-sepharose column. On the other hand, p-AAP-sepharose has no ability to bind to HLADH, although three proteins from a rat liver cytosolic fraction were found to be able to bind to p-AAP-sepharose. CONCLUSIONS: p-HAP binds to ADH in a structure-specific manner and this binding is nonspecific to species of origin of ADH.

Acetaminophen↗
PubMed1975

Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5'-phosphate.

Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage. This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.

Apoenzymes↗
PubMed1986

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts.

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.

Animals↗
PubMed2002

Characterization of virus adsorption by using DEAE-sepharose and octyl-sepharoser.

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and phiX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included phiX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.

Adsorption↗
PubMed1984

Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A.

The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.

Animals↗
PubMed1987

Determination of the leakage from Phenyl-Sepharose Cl-4B, Phenyl-Sepharose FF and Phenyl-Superose in bulk and column experiments.

The release of ligands from Phenyl-Sepharose CL-4B, Phenyl-Sepharose FF and Phenyl-Superose has been studied in bulk and column experiments. The leakage products have been identified and quantified by liquid chromatography, fluorescence spectroscopy and proton NMR spectroscopy. It is demonstrated that the leakage occurs primarily through hydrolysis of the agarose support. However, leakage via ether cleavage of the spacer arm-ligand moiety is also observed especially for Phenyl-Superose. The release of ligands at acidic pH is in agreement with a first-order reaction and correspondingly the rate constants have been estimated for all three gels at pH 1 and 2. These show that 50% of the ligands are intact after 15 years of incubation at pH 2. Phenyl-Superose is the most stable gel at acidic pH, whereas Phenyl-Sepharose CL-4B and Phenyl-Sepharose FF are the most stable at neutral and basic pH.

Chromatography, High Pressure Liquid↗
PubMed1990

[Coupling of bovine gamma globulin to carbonochloridate-activated cellulose beads, CNBr-activated Sepharose CL-4B and NaIO4-oxidized Sepharose 6B and use of the coupled products for immunoaffinity chromatography].

For coupling 25 mg of bovine IgG (BGG) were given to 5 ml volumes of packed bead cellulose activated by 5-norbornene-2.3-dicarboximido carbonochloridate and CNBr-activated Sepharose CL-4B, respectively. Thus, BGG-immunosorbents were obtained with 4.6 to 4.9 mg BGG/ml matrix. 5 ml volumes of packed NaIO4-oxidized Sepharose 6B coupled 27% from 25 mg of BGG, only. In this case, immunosorbents with 1.35 mg BGG/ml matrix were produced. All BGG-immunosorbents were chemically relatively stable. The use of these immunosorbents for affinity chromatography results in the isolation of one milligram of pure rabbit anti-BGG antibodies by means of about 4.6 mg of BGG coupled to the cellulose or the Sepharose-CL-4B matrices. On the other side, only 3.4 mg of BGG coupled to the NaIO4-activated Sepharose 6B were necessary in order to isolate one milligram of antibodies in an immunoelectrophoretically pure state.

Animals↗
PubMed1992

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.

Animals↗
PubMed1982

False RIA IgM titres to herpes simplex virus and cytomegalovirus: factors causing them, and their absorption by protein A-Sepharose/IgG-protein A-Sepharose.

A method for the absorption of false radioimmunoassay (RIA) IgM titres against herpes simplex virus (HSV) and cytomegalovirus (CMV) is presented. The serum specimens were absorbed by a mixture of protein A-Sepharose and protein A-Sepharose saturated with normal human gamma globulin (PAS/IgG). The detection of rheumatoid factor of IgM class (IgM-RF) as well as antinuclear antibodies (ANA) of both IgM and IgG class by solid-phase RIA is also described, and their role in the false IgM results was studied. It was found that the PAS/IgG absorption removed 50-90% of both IgM-RF and total IgG. The reduction of IgM-ANA clustered at 50-90% or nothing, whereas the reduction of IgG-ANA was approximately 50%. The studies with HSV and CMV antigens indicated that the removal of false IgM titres was more effective than the removal of each of these four factors. It was concluded that the IgM-RF titres alone were not sufficiently high to explain the false IgM results, but the ANA activity probably contributed.

Absorption↗
PubMed2006

Non-specific binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be reduced by pre-treatment with glycine or ethanolamine.

Insulin autoantibody (IAA) microassays are widely used for predicting type 1 diabetes. As levels of IAA are often low in type 1 diabetes, non-specific binding (NSB) needs to be minimised if assays are to achieve high analytical sensitivity. IAA microassays use protein A Sepharose (PAS) or protein G Sepharose (PGS) to isolate the antibody-bound label, but NSB by the gel can differ between commercially-produced batches. We investigated whether pre-incubation of gel with glycine or ethanolamine could overcome this problem. Batches of PAS/PGS shown to have high NSB (0.3-3.2%) were incubated with glycine or ethanolamine at various pHs between 8 and 10.6 for 2-18 h at 4 degrees C or room temperature. Treating PAS at pH 10.6 with 0.2 M glycine overnight at room temperature reduced NSB by >84%, with minimal reduction in specific binding (<5%). Treating PGS at pH 10.6 with 0.2 M ethanolamine overnight at 4 degrees C reduced background by >95%, with minimal reduction in specific binding by most sera. Treatment at high pH was critical in reducing NSB to both PAS and PGS, with slight reduction at pH 8, but a major reduction at pH 10.6. Pre-treatment with glycine or ethanolamine allows "poor" batches of PAS or PGS to be used in sensitive IAA assays, improving both consistency and performance.

Autoantibodies↗