Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.     

Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.     

The effect of matrix on the binding of albumin to immobilized Cibacron Blue.

With Cibacron Blue as a ligand, the suitability of Sepharose, Sephacryl, cellulose and Ultrogel as column supports for the affinity chromatography of human serum albumin was examined. Sepharose and Sephacryl were the most effective matrices. Albumin was readily removed from such columns by using an electrophoretic desorption technique.     

Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand.     

Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase

Protein-protein interactions between cytochrome P450 (P450) and other drug-metabolizing enzymes were studied by affinity chromatography using CYP1A1-, glycine-, and bovine serum albumin (BSA)-conjugated Sepharose 4B columns. Sodium cholate-solubilized microsomes from phenobarbital-treated rat liver were applied to the columns and the material eluted with buffer containing NaCl was analyzed by immunoblotting. Microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferases (UGTs), as well as NADPH-P450 reductase, were efficiently trapped by the CYP1A1 column. Glycine and BSA columns exhibited no ability to retain these proteins. Protein disulfide isomerase and calnexin, non-drug-metabolizing enzymes expressed in the endoplasmic reticulum, were unable to associate with the CYP1A1 column. These results suggest that CYP1A1 interacts with mEH and UGT to facilitate a series of multistep drug metabolic conversions.     

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives.

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.     

Purification of cofactor-dependent enzymes by affinity chromatography

Several 8-(6-aminohexyl)-amino adenine nucleotide derivatives, including ATP, 2′,5′-ADP, 3′,5′-ADP and desulfo-CoA (CoA, reduced coenzyme A), were prepared and immobilized on Sepharose by cyanogen bromide activation. 8-(6-Aminohexyl)-amino-ATP-Sepharose was found to exhibit good affinity for both NAD⁺-dependent dehydrogenases and kinases. Sequential biospecific elutions with NADH and ATP resulted in a good separation of dehydrogenases from kinases. As many as eight different dehydrogenases and kinases could be substantially purified from both porcine muscle and mouse kidney extracts by this new procedure. 8-(6-Aminohexyl)-amino-2′,5′-ADP- and −3′,5′-ADP-Sepharose were shown to exhibit good affinity for many NADP⁺-dependent dehydrogenases from yeast extracts and CoA-dependent enzymes, respectively. Purification of citrate synthases from pig heart and Eschericia coli extracts by means of these 8-substituted adenine nucleotide affinity columns was also presented.     

The synthesis of 8-(6-aminohexyl)-amino-GTP and -GDP and their application as ligands in affinity chromatography

Two guanine nucleotide analogs, 8-(6-aminohexyl)-amino-guanosine-5′-triphosphate and 8-(6-aminohexyl)-amino-guanosine-5′-diphosphate, were synthesized from the monophosphate by phosphorylation with pyrophosphate or orthophosphate. Structural assignments were made according to nuclear magnetic resonance spectra. The ability of these nucleotides to act as enzyme substrates has been determined. Both guanosine nucleotides were used as ligands for affinity chromatography by attaching them to Sepharose 4B by the cyanogen bromide method. The effectiveness of these new affinity columns in enzyme purification was investigated with polypeptide elongation factor II from rat and pig liver.     

Rapid purification by affinity chromatography of rat brain pyridoxal kinase and pyridoxamine-5-phosphate oxidase

Rat brain pyridoxal kinase and pyridoxamine 5′ phosphate oxidase have been purified to electrophoretic homogeneity using pyridoxyl Sepharose and phosphopyridoxyl Sepharose columns as the first stages in the purification procedures. These affinity supports were synthesized by two simple steps consisting of reacting pyridoxal and pyridoxal 5′ phosphate with commercially available ω-aminohexyl Sepharose and subsequent reduction of the resultant Schiff's bases with sodium borohydride. This method allows total purification of both enzymes from the same tissue source in 4–5 days.     

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-purification of coenzyme-dependent enzymes by affinity chromatography

Recent developments in general ligand affinity chromatography have greatly facilitated the purification of numerous coenzyme-dependent enzymes in practical scale. By immobilizing various derivatives of coenzymes (NAD+, NADP+, ATP and coenzyme A) on Sepharose, these general ligand affinity columns have the potential of enriching as many as 30% of the two thousand known enzymes as well as their isozymic forms, if they are applied in combination with the conventional procedures.     

Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography

The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr-activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.     

Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked sepharose: An efficient electrophoretic elution procedure

An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ～ 10¹⁰ M^?1) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ～ 10¹⁰ M^?1) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH₄OH.The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ～ 10¹¹ M^?1). On the other hand the residual 40% fraction of antibodies resistant to NH₄OH elution was denaturated.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

High performance affinity chromatography of Bacillus neutral proteases.

Bacillus neutral proteases were purified using bacitracin-silica as an affinity medium. Several chromatographic procedures were investigated, including high speed runs on columns with 40- to 60-microns silica particles. The high speed procedure enabled the purification of 4.9 mg of B. subtilis neutral protease directly from 165-ml culture supernatant within 1.5 h. The neutral proteases of B. polymyxa and B. stearothermophilus were also purified. The latter enzyme was further concentrated by a second affinity chromatography step, using Sepharose with glycyl-D-phenylalanine as a ligand. During the purification procedures isopropanol was used to prevent autodigestion of the enzymes.     

Affinity chromatography of nicotinamide nucleotide-dependent dehydrogenases on immobilized nucleotide derivatives

A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for `general ligand' affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD⁺ and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD⁺ to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Inositol 1,4,5-trisphosphate affinity chromatography

Inositol 1,4,5-trisphosphate (IP3) affinity columns were made by coupling IP3 analogs to a supporting matrix. Sepharose 4B. IP3 5-phosphatase activity. IP3 3-kinase activity and IP3 binding activity from rat brain were absorbed on the IP3 columns. and were eluted by increasing KC1 concentration. This purification procedure increased the specific activities of these parameters 5-200-fold. Thus Sepharose 4B immobilized IP3 analogs can specifically interact with IP3-binding proteins, demonstrating that IP3 affinity columns are a good method for purifying such proteins. Furthermore, our results suggest that IP3 analogs can be linked to other molecules to make useful derivatives without loss of their biological activities.