Blue dextran-Sepharose Chromatography Research Articles

CAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina.

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells.

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NCR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NCR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heme-iron-containing (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NCR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

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.

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.

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.

Synthesis of Nitrate Reductase in Chlorella

Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically.

Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography.

The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.

Interaction of Cibacron Blue F3GA with Escherichia coli DNA polymerase I and with T4 DNA polymerase

Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E.coli: a potential application for the purification of the enzyme

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.

A rapid purification of T4 polynucleotide kinase using blue dextran-sepharose chromatography

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5′-dephosphopolynucleotide 5′-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactivity labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.

D-3-Phosphoglycerate dehydrogenase from chicken liver. I. Purification.

A method is described for the preparation of homogeneous D-3-phosphoglycerate dehydrogenase from chicken liver in amounts sufficient for structural studies. The procedure utilizes ammonium sulfate precipitation, blue dextran-Sepharose chromatography, ion exchange chromatography on phosphocellulose, and crystallization. Previous reports of instability of the enzyme have been shown to be due to proteolysis in the crude extract which can be effectively prevented by leupeptin. The purified enzyme is a basic protein with a pI of 8.95 as measured by isoelectric focusing. The extinction coefficient at 278 nm of a 1% solution is 5.3.

Partial purification of the chick intestinal receptor for 1,25-dihydroxy vitamin D by ion exchange and blue dextran–sepharose chromatography

Partial purification of the chick intestinal receptor for 1,25-dihydroxy vitamin D by ion exchange and blue dextran–sepharose chromatography

Proposed structure for brain adenylate cyclase purified using blue dextran–Sepharose chromatography

WE have recently shown1 that blue dextran–Sepharose affinity chromatography can be used to advantage to abbreviate markedly the purification procedures for soluble enzymes whose nucleoside phosphate binding sites are constructed by a supersecondary structure called the NAD-domain. Preliminary measurements using lactate dehydrogenase indicated that affinity chromatography with blue dextran–Sepharose can also be done in the presence of various non-ionic detergents used to solubilise membrane proteins. Here we describe the use of blue dextran–Sepharose affinity chromatography for the purification of the membrane-bound hormone-responsive enzyme, adenylate cyclase, and propose a new structure for the membrane complex which does not contain a separate hormone receptor protein.

A quantitative procedure for recovery of nucleoside phosphate ligands and preparation of apoprotein from holoproteins using blue dextran-Sepharose chromatography

A simple chromatographic procedure is described for removal of strongly bound ligands from holoproteins whose ligand sites are constructed by an NAD-like domain. Application of such a holoprotein solution to a blue dextran-Sepharose chromatographic column equilibrated with a low ionic strength solvent results in recovery of the ligand and retention of the apoprotein. The apoprotein is then recovered by either batch or gradient elution with solvents having a high ionic strength. The quantitative removal of NAD from glyceraldehyde-3-phosphate dehydrogenase and the preparation of its apoprotein are illustrated.

Studies on cyclic GMP-dependent protein kinase properties by blue dextran-sepharose chromatography

Cyclic GMP-dependent protein kinase prepared from calf lung was studied for its binding properties with blue dextran-Sepharose affinity column chromatography. Blue dextran competitively inhibited [ 3 H]cGMP binding to the enzyme. ATP + Mg ++ did not prevent cGMP-kinase binding to blue dextran, nor did it facilitate the liberation of blue dextranbound enzyme. Substrate proteins such as histone and protamine dissociated the native enzyme into subunits. Considering all these results, cGMP-kinase seemed to conform with the “dissociation model” proposed for cAMP-kinase but with peculiarities of binding to blue dextran.