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PubMed1986

Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin.

The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.

Animals↗
PubMed1978

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.

Acid Anhydride Hydrolases↗
PubMed1999

Immobilized 4-aminophenyl 1-thio-beta-D-galactofuranoside as a matrix for affinity purification of an exo-beta-D-galactofuranosidase.

An alternative and fast method for the purification of an exo-beta-D-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-beta-D-galactofuranoside affinity chromatography system and specific elution with 10 mM D-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM D-galactono-1,4-lactone in a 100-500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-beta-D-galactofuranosidase was ascertained through binding to Concanavalin A-Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and Km and Vmax values of 0.311 mM and 17 mumol h-1 microgram-1 respectively, when 4-nitrophenyl beta-D-galactofuranoside was employed as the substrate.

Chromatography, Affinity↗
PubMed2003

Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase.

Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives. The observed interaction with cytokinins is discussed in view of a putative role for adenosine kinase in TBY-2 cytokinin metabolism. The presented results show for the first time a plant adenosine kinase affinity-purified to homogeneity that was identified by primary structure analysis.

Adenosine Kinase↗
PubMed1992

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Animals↗
PubMed1976

Acetylcholinesterase of the house-fly head. Affinity purification and subunit composition.

1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.

Acetylcholinesterase↗
PubMed1993

One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.

We have previously purified both shiga-like toxin (SLT) I and II using the toxins' affinity to P1 glycoprotein (P1gp) from hydatid cyst material (HCM). Binding of these toxins is based on their affinity for terminal Gal alpha 1-->4Gal disaccharide residues present in HCM. Although the binding specificity of SLT-II variants (v) differs from that of STL-II they are reported to recognize Gb3 and should bind to P1gp. Therefore we examined the usefulness of HCM to purify SLT-IIv of porcine (p) and human (h) origin. Toxins were purified from fermenter culture supernatants of Escherichia coli HB101 (pDLW5) (SLT-IIvp), and E. coli DH5 alpha (pJES210) (SLT-IIvh) utilizing HCM. SLT-IIvh and SLT-IIvp consisted of A and B subunits, as determined by SDS-PAGE. We obtained 0.16 mg SLT-IIvp and 0.12 mg SLT-IIvh/I of culture (yields > 65%). Various capture systems to detect shiga toxin, SLT-II, SLT-IIvp and SLT-IIvh by ELISA were examined. All toxins bound to HCM, and all except SLT-IIvp bound to the monoclonal antibody 4D1. Only SLT-IIvp bound to the glycolipid Gb4, and only shiga toxin bound significantly to Gb3. Similarities in the level of Gb4 expression in HeLa 229 (ATCC) and Vero cells may explain the lack of differential cytotoxicity between SLT-IIvp and SLT-IIvh on these cell lines.

Animals↗
PubMed1994

Affinity purification of Hydra glutathione binding proteins.

The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH-binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity-purified proteins was a triplet of peptides ranging in molecular weight from 24.5-26 kDa. Antiserum generated against the 24.5-26 kDa triplet peptides inhibited GSH-stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.

Animals↗
PubMed2002

Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with protein A and protein G.

This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.

Adaptor Proteins, Signal Transducing↗
PubMed1990

A novel affinity purification of D-1 dopamine receptors from rat striatum.

When rat striatal membranes were pretreated with the sulfhydryl (-SH) modifying reagent, N-ethylmaleimide (NEM) in the presence of the D-1-specific agonist, SKF R-38393, the D-1 dopamine receptor was completely protected from NEM-mediated inactivation. The D-1 receptors, solubilized from these membranes with 1% sodium cholate in the presence of phospholipids, bound with high efficiency (greater than 90%) to mercury-agarose columns. The bound receptors were eluted from the affinity column with a -SH reducing agent, beta-mercaptoethanol. Upon removal of beta-mercaptoethanol from the eluted fractions by inclusion chromatography, the receptor was reconstituted into phospholipid vesicles and assayed for ligand binding activity. The affinity purified receptor exhibited saturable and specific binding of the D-1-specific ligand 125I-SCH 23982, with a Kd of 1.6 nM comparable to that measured in intact membranes and solubilized extracts. The binding capacity of these receptors for 125I-SCH 23982 was 11,000 pmol/mg protein, representing greater than an 8000-fold purification over the starting membrane preparation. The purity of the affinity eluted receptors was estimated to be 78%. The purified receptors retained the pharmacological properties of membrane-bound receptors, including the ability to distinguish between active and inactive enantiomers of specific dopaminergic antagonists. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining revealed the presence of two major polypeptides of 74 and 54 kDa. These two polypeptides were absent in those affinity eluted fractions which did not display 125I-SCH 23982-binding activity and also were not detected in preparations obtained from membranes which were NEM-treated in the absence of D-1-specific agonist. The molecular weights of these polypeptides were similar to those of membrane-bound D-1 receptors, when labeled with a D-1-specific photo-affinity ligand, 125I-8-hydroxy-3-methyl-1-(4-azidphenyl)-2,3,4,5-tetrahydro-1H-3-b enzazepine. These two polypeptides may represent glycosylated and deglycosylated forms of the D-1 dopamine receptor.

Animals↗
PubMed2003

Affinity purification of specific chromatin segments from chromosomal loci in yeast.

Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states.

Chromatin↗
PubMed1997

Affinity purification and characterization of glucosidase II from pig liver.

Glucosidase II has been purified from crude pig liver microsomes by a convenient procedure involving DEAE-Sephacel, Con A-Sepharose and affinity chromatography on N-5-carboxypentyl-1-deoxynojirimycin-AH-Sepharose. Specific binding of glucosidase II to the affinity matrix required its prior separation from glucosidase I, which was accomplished by fractional Con A-Sepharose chromatography. The three-step procedure yielded, with approximately 15% enzyme recovery, a > 190-fold enriched glucosidase II, consisting of two proteins (107 kDa and 112 kDa). Both polypeptides are N-glycosylated with probably one glycan chain, in line with their binding to Con A-Sepharose. Immunological cross-reactivity and other experimental data indicate that the 107 kDa N-glycoprotein is derived from the 112 kDa species by partial proteolysis. The occasional presence of a 60 kDa peptide co-eluting with the catalytic activity suggests that glucosidase II may be associated with other protein subunit(s) in a heteromeric membrane complex. Glucosidase II hydrolyzes the alpha1,3-glucosidic linkages in Glc(2-1)-Man9-GlcNAc2, as well as synthetic alpha-glucosides, efficiently but does not remove the distal alpha1,2-linked glucose in Glc3-Man9-GlcNAc2. The enzyme has a pH optimum close to 6.5 and is not metal ion-dependent. Catalytic activity is strongly inhibited by basic sugar analogues including 1-deoxynojirimycin (dNM; app. Ki approximately 7.0 microM), N-5-carboxypentyl-dNM (app. Ki approximately 32 microM) and castanospermine (app. Ki approximately 40 microM). Substitution of the 3-OH or 6-OH group in dNM by a fluoro group reduces the inhibitory potential drastically. We conclude from these observations that the two hydroxy groups are essential for inhibitor/substrate binding due to their ability to interfere as hydrogen bond donors. A polyclonal antibody raised against the 107 kDa polypeptide reacted specifically with two proteins from different cell types on Western blots. Their molecular masses were identical with those from pig liver microsomes, pointing to a highly conserved amino acid sequence of glucosidase II. This suggests that the variance in molecular mass for glucosidase II reported for the enzyme from other tissues and species may be due to partial proteolysis.

Animals↗
PubMed2006

Affinity purification and partial characterization of IgM-like immunoglobulins of African catfish, Clarias gariepinus (Burchell, 1822).

IgM like macroglobulin from bovine serum albumin (BSA)-immunized African catfish C. gariepinus was purified by affinity chromatography and partially characterized. The molecular weight of this macroglobulin was 840 kDa, as estimated by gel filtration chromatography. Purified macroglobulin was analyzed using SDS-PAGE under reducing and non-reducing conditions. The molecular weight (MW) of heavy and light chain was 74.8 kDa and 27.2 kDa respectively, in presence of a reducing agent. In non-reducing SDS-PAGE, a single high MW band was observed representing tetrameric form.

Animals↗
PubMedDate not supplied

Review of conformation-specific affinity purification methods for plasma vitamin K-dependent proteins.

There are seven known vitamin K-dependent proteins in blood. These proteins require calcium ion for expressing their full biological activities. Calcium ion also induces conformational changes in this class of proteins. Taking advantage of the ligand induced conformational changes, a number of unique approaches of affinity chromatography have been developed. These methodologies have been very useful tools for both the purification and for understanding the structure-function relationships of this class of proteins. One method is the use of metal ion dependent immunoaffinity chromatography. The antigen can be dissociated from the antibodies with either the removal or addition of calcium ion under physiological conditions. The other method is pseudoaffinity chromatography. This method uses conventional ion-exchange or hydrophobic resin and manipulates the mobilities of the proteins on these resins by the presence or absence of calcium ions. Researchers working with other calcium binding proteins or other proteins that are known to undergo ligand induced conformational changes may benefit from the experience of these unique conformation-specific affinity chromatography approaches.

1-Carboxyglutamic Acid↗
PubMed1998

Affinity purification of the Ca-ATPase from cardiac sarcoplasmic reticulum membranes.

We report the isolation of the functional form of the Ca-ATPase from porcine cardiac sarcoplasmic reticulum (SR) membranes, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Red 120. Conditions that optimize the solubility and functional stability of the cardiac Ca-ATPase in detergent during the purification procedure are essential to its recovery. The purified Ca-ATPase migrates as a single band on Coomassie blue-stained polyacrylamide gels and exhibits high specific activity (2.5 IU at 25 degreesC) and functional stability. Similar enrichment of the Ca-ATPase estimated from either relative amounts of the 100-kDa protein band on polyacrylamide gels or steady-state concentrations of phosphorylated enzyme intermediate (E-P) demonstrate that neither nonfunctional Ca-ATPases nor non-Ca-ATPase proteins migrating with an apparent molecular weight of 100 kDa constitute a significant fraction of these preparations. Steady-state levels of E-P are 1.3 and 8.6 nmol/mg protein, respectively, for native cardiac SR membranes and the final purified fraction. These values, in comparison to the maximum value (9.1 nmol/mg) for the 110-kDa protein, agree well with estimates of total Ca-ATPase abundance from gel densitometry for both preparations and indicate full site reactivity, i.e., one phosphorylation site for each 110-kDa cardiac Ca-ATPase polypeptide chain.

Animals↗
PubMed1987

Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody.

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.

Antibodies↗