Blue dextran-Sepharose Research Articles

Purification and specificity of RNase A3 from Vicia faba root cells

Vicia faba root ribonucleases are bound to Cibacron blue F3GA. A Blue dextran-Sepharose column was used to purify RNase A3, the more abundant enzyme from V. faba root. Using dinucleoside monophosphate as substrates, it appears that this enzyme behaves as a cyclizing phosphotransferase. With high enzyme/substrate ratios on prolonged digestion a partial release of a nucleoside 3′ phosphate occurs. The specificity is relatively high since only the purine-purine phosphodiester linkages out of 16 types of possible links are easily cleaved. When a pyrimidine is involved in the phosphodiester bond, a much slower rate of attack (Py in 5′) or no attack (Py in 3′) was detected.

Purification and specificity of RNase A 3 from Vicia faba root cells

Vicia faba root ribonucleases are bound to Cibacron blue F3GA. A Blue dextran-Sepharose column was used to purify RNase A 3, the more abundant enzyme from V. faba root. Using dinucleoside monophosphate as substrates, it appears that this enzyme behaves as a cyclizing phosphotransferase. With high enzyme/substrate ratios on prolonged digestion a partial release of a nucleoside 3′ phosphate occurs. The specificity is relatively high since only the purine-purine phosphodiester linkages out of 16 types of possible links are easily cleaved. When a pyrimidine is involved in the phosphodiester bond, a much slower rate of attack (Py in 5′) or no attack (Py in 3′) was detected.

A monoisozymic nucleoside diphosphate kinase capable of complete phosphorylation.

Nucleoside diphosphate kinase (EC 2.7.4.6) has been purified nearly 2000-fold from beef brain particulate material in 65% yield. The purification procedure described here takes advantage of the relatively unique complexation of the enzyme with blue dextran-Sepharose in the presence of a nonionic detergent. Unlike preparations of the enzyme previously described, the bovine brain enzyme is monoisozymic and each polypeptide chain can be phosphorylated by a nucleoside triphosphate substrate. Catalytically functional forms of the enzyme having lower pI values can be generated by proteolysis.

The effect of proteolysis on the calmodulin activation of cyclic nucleotide phosphodiesterase.

A high Km cytoplasmic cyclic nucleotide phosphodiesterase (EC 3.4.1.17) has been obtained from bovine brain. The unproteolyzed enzyme contains (63 +/- 1) X 10(3) molecular weight polypeptide chains which exhibit little if any basal catalytic activity. Complexation with calmodulin stimulates the catalytic activity nearly 2 orders of magnitude, presumably, by causing a conformational change in the enzyme which either creates or exposes the catalytic sites. Removal of about 120 amino acids from the terminal portion(s) of each polypeptide chain either by an endogenous protease or by exogenous trypsin prevents calmodulin complexation and generates a basal catalytic activity equivalent to that of the unproteolyzed enzyme-calmodulin complex. In contrast to affinity chromatography using immobilized calmodulin, blue dextran-Sepharose chromatography can be used to select for enzyme containing only unproteolyzed polypeptide chains.

Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure.

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.

Rabbit liver fructose 1,6-bisphosphatase: Labeling of the active and allosteric sites with pyridoxal 5-phosphate and sequence of a nonapeptide from the active site

The active and allosteric sites of fructose 1,6-bisphosphatase (Fru-P 2ase, EC 3.1.3.11) were labeled by reaction with pyridoxal phosphate and sodium borohydride in the presence of the allosteric inhibitor AMP or the substrate, Fru-P 2, respectively. Modification of the active site results in loss of activity. Modification of the allosteric site decreases the sensitivity of the enzyme to inhibition by AMP and alters its ability to bind to blue dextran-Sepharose. The allosteric and active sites have been located on different cyanogen bromide peptides; the sequence of a nonapeptide from the active site is (H)GlyLysLeuArgLeuLeu TyrGluCys(OH). The lysyl residue is modified by pyridoxal phosphate.

Identification of an Epstein-Barr virus nuclear antigen by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis.

A 65,000-dalton (65K) antigen found in Raji cells by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis has been identified as an Epstein-Barr virus nuclear antigen (EBNA). This identification is based on the following evidence. The 65K antigen is detected in Raji cells but not in three Epstein-Barr virus (-) human B cell lines. It is not detected with EBNA (-) sera. The 65K antigen is found predominantly in the nucleus and co-elutes with EBNA during partial purification by DNA-Sepharose and Blue Dextran-Sepharose chromatography. Finally, the partially purified 65K antigen is an effective absorbant of EBNA antibody as measured in an anticomplement immunofluorescence assay. Antigens with molecular weights of 72, 70, and 73K have been detected in B95-8, P3HR-1, and Namalwa cells, respectively. These antigens are the likely homologues of the 65K Raji EBNA. In addition, an Epstein-Barr virus-associated, 81K DNA-binding antigen has been detected in both B95-8 and Raji cells.

A high-field purification of glyoxalase I from rabbit liver by affinity chromatography on Blue Dextran-Sepharose 4B

An improved method is described for the purification of glyoxalase I from rabbit liver. The method involves homogenization, ammonium sulfate precipitation followed by chloroform/ethanol precipitation, affinity chromatography on Blue Dextran-Sepharose 4B and chromatography on Sephadex G-100. The enzyme is specifically eluted from the affinity column by S-hexylglutathione, a competitive inhibitor of the enzyme. This procedure offers a convenient method for obtaining electrophoretically pure glyoxylase I in high yields (60–70%).

Purification and molecular characterization of human fibroblast interferon

Human fibroblast interferon was purified from serum-containing culture medium by a combination of concanavalin A or Blue Dextran Sepharose affinity chromatography with high-performance liquid chromatography to material exhibiting a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interferon could be chromatographed and purified at acidic pH in volatile buffers on RP-8, RP-18, cyclohexyl, phenylalkyl, diphenyl, cyanopropyl, and diol supports. A specific activity averaging around 4 × 10 8 units/mg was found for the pure material with a molecular weight of 20,000–21,000 after 20,000- to 50,000-fold purifications. In some preparations, low activity levels were also found at positions corresponding to 10,000, 17,000–18,000, 35,000, and 40,000 daltons. Amino acid and amino sugar analysis, partial NH 2- and COOH-terminal sequences, and tryptic peptide patterns determined at the picomole level are reported for the purified interferon.

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12.

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.

Isolation of two species of human lymphoblastoid (Namalva) cell-derived interferon that differ in rates of clearance, size, and cell specificity.

Chromatography of human lymphoblastoid (Namalva) interferon on blue dextran Sepharose has separated the interferon activity into two species which differ in affinity to the dye Cibacron Blue F3GA. The separated species differ also in rates of clearance from the circulation of mice, size, and specificity toward human and bovine cells. The species retained by the dye manifests a slower clearance rate, is about 20,000 mol. wt., and is equally active on both human fibroblasts FS-11A and bovine cell line MDBK. The unretained species disappears from the circulation at a faster rate, is 16,000 mol. wt., is active on bovine cells, but almost inactive on human cells. It is suggested that a comparative study of the two species may aid in the elucidation of the causes responsible for the rapid clearance or bioinactivation of interferon.

Affinity of Leuconostoc phosphoglycerate mutase for blue dextran-sepharose.

Phosphoglycerate mutases (EC 2.7.5.3) of several lactic acid bacteria were adsorbed to blue dextran-Sepharose 4B and eluted quantitatively with 1 M KC1. Phosphoglycerate mutase of Leuconostoc dextranicum AHU 1078 was eluted with 3-phosphoglycerate, 2-phosphoglycerate or 2,3-bisphosphoglycerate at 1 mM. Furthermore, ATP, ADP, GTP and GDP were also effective for eluting the mutase from the blue dextran-Sepharose column, but AMP and GMP were not. Although NADP(H) eluted the mutase, NAD(H) did not. By single step purification using linear gradient elution of 2,3-bisphosphoglycerate between 0 and 0.5 mM, the Leuconostoc mutase was purified to electrophoretic homogeneity with a 62.5% yield. The difference in spectrum of Cibacron Blue in the presence of the mutase was quite similar to that of the dye in the presence of KC1, but not in the presence of glycerol. The Leuconostoc mutase was inactivated by pyridoxal phosphate. This inactivation was prevented in the presence of blue dextran, as well as 3-phosphoglycerate.

Interaction of phosphoglycerate kinase from Escherichia coli with cibacron blue.

Phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) from Escherichia coli AHU1520 was purified until electrophoretically homogeneous by a single procedure, using a linear gradient elution of 2, 3-bisphosphoglycerate between 0 and 0.5 mM on a blue dextran-Sepharose 4B column chromatography. The phosphoglycerate kinase was inhibited by blue dextran competitively with respect to 3-phosphoglycerate, and noncompetitively with respect toATP. The inhibition of the kinase by Cibacron Blue was competitive with respect to 3-phosphoglycerate at both saturated and unsaturated levels of ATP; the inhibition by the dye toward ATP was shifted from a mixed-type at a saturated level of 3-phosphoglycerate to competitive-type at an unsaturated level. Further inhibition studies implied that two molecules of the dye were bound per molecule of phosphoglycerate kinase with an unsaturated level of ATP used as a fixed substrate. The shape of the difference spectrum of Cibacron Blue in the presence of the kinase was very similar to that of a difference spectrum generated upon addition of KCl, but was markedly different from the shape of the spectrum produced by the addition of glycerol.

63] Purification of human fibroblast interferon by high-performance liquid chromatography

This chapter presents a procedure combining the techniques of affinity chromatography and high-performance liquid chromatography (HPLC) for the preparation of fibroblast interferon from serum-containing medium. The purified fibroblast interferon is homogeneous by several criteria of molecular characterization and has a specific activity of approximately 4 x l0 8 units/mg. The purified protein is present in a completely volatile, detergent-free solvent. From several possible approaches, the combination of Blue Dextran-Sepharose affinity chromatography with reverse-phase HPLC at acidic pH is outlined. In contrast to leukocyte interferon, it is not possible to use neutral pH conditions for the HPLC steps because fibroblast interferon rapidly lost activity when organic solvents (propanols, acetone, Methylcellosolve, and such) are present. The data for the homogeneous protein is a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which coincided with the recoverable antiviral activity, and a single protein peak in the last chromatographic step, which coincided with the antiviral activity. The purified preparations are characterized by amino acid analyses, amino sugar analyses, end-group determinations, tryptic maps, and amino acid sequencing.

Purification and peptide mapping of rat brain choline acetyltransferase.

Acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6) has been purified approximately 17,000-fold from rat brain. The purification protocol included acid and NH4SO4 precipitation, followed by chromatography on CM-Sephadex, phenyl-Sepharose, Sephadex G-150, and blue dextran Sepharose. Two peaks of enzyme activity were detected after blue dextran Sepharose chromatography containing an overall yield of approximately 4% of the starting enzyme activity. The final specific activity of the blue dextran fractions was 70 to 80 mumol of acetylcholine formed min-1 mg of protein-1. Sodium dodecyl sulfate electrophoresis of the blue dextran fractions revealed three closely spaced proteins with a molecular weight of approximately 67,000. Tryptic peptide maps were prepared for each of the gel bands and indicated that they contained nearly identical primary structure.

Interaction of yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase with Blue-Dextran sepharose: Assignement of the Blue-Dextran binding site on the synthetases

Yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase like nucleotidyl transferases previously investigated interact with the Blue-Dextran-Sepharose affinity ligand through their tRNA binding domain: the enzymes are readily displaced from the affinity column by their cognate tRNAs but not by ATP or a mixture of ATP and the cognate amino acid in contrast to other aminoacyl-tRNA synthetases. In the absence of Mg ++, the arginyl-tRNA synthetase can be dissociated from the column by tRNA Asp and tRNA Phe which have been shown to be able to form a complex with the synthetase, but in presence of Mg ++ the elution is only obtained by the specific tRNA. The procedure described here can thus be used: (i) to detect polynucleotide binding sites in a protein; (ii) to estimate the relative affinities of different tRNAs for a purified synthetase; (iii) to purify an aminoacyl-tRNA synthetase by selective elution with the cognate tRNA.

Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase.

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].

Substrate-induced dissociation of glyceraldehyde-phosphate dehydrogenase detected by affinity chromatography. Study of subunit interactions by affinity sorption

All four NAD-binding domains of the tetrameric apoglyceraldehyde-phosphate dehydrogenase ( d-glyceraldehyde 3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) displayed identical affinities ( K d = 4.8 · 10 −5 M ) towards the chromophore of Blue Dextran. The enzyme species which contained only the most firmly bound NAD coupled the dye through the remaining free sites with the same affinity as the apoenzyme. The interaction of glyceraldehyde-phosphate dehydrogenase and Blue Dextran-Sepharose was characterized by the aid of an affinity sorbent batch technique. We found that the apparent dissociation constant varied with enzyme concentration showing preferential binding in dilute solutions. This suggests that the monomeric and/or dimeric form(s) of glyceraldehyde-phosphate dehydrogenase have higher affinity towards the sorbent than the tetramer. The apparent K d of the enzyme-Blue Dextran-Sepharose complex at low enzyme concentration was similar to the K i of the competitive inhibition of enzyme activity by free Blue Dextran, i.e. about 10 −6 M. Glyceraldehyde 3-phosphate increased the binding of the enzyme to the immobilized dye in chromatographic experiments and decreased the apparent dissociation constant in the batch system if enzyme solutions of around 10 −6 M were examined. In more dilute solutions glyceraldehyde 3-phosphate had no effect on complex formation. These data suggest that glyceraldehyde 3-phosphate influences the interaction of the enzyme and the immobilized dye, first of all, by shifting the equilibrium of the different enzyme forms towards dissociation.

A one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver

Adsorption to blue dextran-Sepharose and successive elution with AMP and fructose 1,6-bisphosphate provides a one-step procedure for the simultaneous purification of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver homogenates. The procedure can be completed in a single day and the recoveries are approximately 40–50% for each enzyme activity. When the procedure is applied to a mixture of the purified enzymes, the recoveries are nearly quantitative.

Purification and characterization of a poly(A) polymerase from beef liver nuclei.

Poly(A) polymerase [EC 2.7.7.19] was highly purified from beef liver nuclei by the use of column chromatographies on heparin-Sepharose 4B and Blue Dextran-Sepharose 4B. The purified enzyme showed one major protein band of the molecular weight of 57,000 in SDS polyacrylamide gel electrophoresis, which agreed with the molecular weight estimated from glycerol gradient centrifugation. The enzyme required the presence of Mn2+ for its activity but was almost completely inactive with Mg2+. It incorporated specifically ATP into polynucleotide as a sole substrate. The enzyme activity dependend entirely on the addition of exogenous polynucleotide primer. It showed certain selectivity for the primers. The most effective among the tested polynucleotides was a short poly(A), for which the Km of the enzyme was shown to be 7 microM. Poly(G, U) and short poly(U) also primed the reaction, but tRNA, phage RNA, poly(G), and poly(C) were inactive. Based on observed specificity for the primer, the role of this enzyme in the cell nuclei was discussed. Digestion of the reaction product of this enzyme by two specific exonucleases, snake venom and spleen phosphodiesterases, suggested that this enzyme catalyzed the covalent bonding of the substrate to the 3' terminus of the primer as in the manner expected for in vivo polyadenylation.