Blue dextran-Sepharose 4B Research Articles

Affinity adsorption and hydrodynamic behavior in a tapered-bed of upward flow

Affinity chromatography operated in a tapered-bed with the continuous up-flow was researched. Purification of Lysozyme by Blue Dextran Sepharose 4B was chosen as a model system. A series of experiments such as the batchwise adsorption, tapered columnwise adsorption, elution and pressure drop determination were carried out to find out characteristics such as adsorption equilibrium, hydrodynamic performance and adsorption efficiency for this kind of new affinity process. A mathematical model to describe this system was also established and verified. Research results showed that the affinity purification by the tapered-bed with up-flow could be operated in three regimes, i.e. the fixed-bed regime, fully fluidized-bed regime and partially fluidized-bed regime, which depended on the flow rate. The adsorption efficiencies were the highest in the fixed-bed regime, and poor adsorption efficiencies were shown in the fully fluidized-bed regime.

Crystallization and Properties of NADPH-Dependent L-Sorbose Reductase from Gluconobacter melanogenus IFO 3294.

NADPH-Dependent L-sorbose reductase (SORD, synonimously NADP-dependent D-srobitol dehydrogenase) was purified and crystallized for the first time from the cytosolic fraction of Gluconobacter melanogenus IFO 3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran Sepharose 4B column was effective for purifying the enzyme giving about 770-fold purification with an overall yield of more than 50%. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.8 s. Gel filtration on a Sephadex G-75 column gave the molecular mass of 60 kDa to the enzyme, which dissociated into 30 kDa subunit on SDS-PAGE, indicating that the enzyme is composed of 2 identical subunits. Reduction of L-sorbose to D-sorbitol predominated in the presence of NADPH with the optimum pH of 5.0-7.0. Oxidation of D-sorbitol to L-sorbose was observed in the presence of NADP at the optimum pH of 7.0-9.0. The relative rate of L-sorbose reduction was more than seven times higher to that of D-sorbitol oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction in the presence of NADPH did not occur with SORD. Since the reaction rate in L-sorbose reduction highly predominated over D-sorbitol oxidation over a wide pH range, the enzyme could be available for direct enzymatic measurement of L-sorbose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the L-sorbose reduced. Judging from the enzymatic properties, SORD would contribute to the intracellular assimilation of L-sorbose incorporated from outside the cells where L-sorbose is accumulated in huge amounts in the culture medium.

Purification and properties of rat liver peroxisomal very-long-chain acyl-CoA synthetase.

It is generally accepted that there are two different acyl-CoA synthetases in rat liver peroxisomes. One is long-chain acyl-CoA synthetase, and the other very-long-chain acyl-CoA synthetase. Nowadays, the nature of long-chain acyl-CoA synthetase is well-known, but that of very-long-chain acyl-CoA synthetase remains unclear. Very-long-chain acyl-CoA synthetase has been extracted from the washed membrane fraction of frozen rat liver peroxisomes with a buffer containing a detergent, and has been purified by chromatography on Ultrogel AcA 34, calcium phosphate gel/cellulose, blue dextran-Sepharose 4B and DEAE-Toyopearl. The molecular masses of the native enzyme and the subunit were estimated to be 235 and 70 kDa, respectively. This enzyme showed marked differences in behavior from long-chain acyl-CoA synthetase during purification. The carbon chain length specificity of very-long-chain acyl-CoA synthetase differed from that of long-chain acyl-CoA synthetase. Very-long-chain acyl-CoA synthetase was active toward long- and very-long-chain fatty acids, but more active toward very-long-chain fatty acids compared with long-chain acyl-CoA synthetase. Antibodies against long-chain acyl-CoA synthetase did not cross-react to very-long-chain acyl-CoA synthetase. Based on these data, the final enzyme preparation is judged to be highly purified very-long-chain acyl-CoA synthetase from rat liver peroxisomes.

Interaction between cytosolic adenylate kinase and nicotinamide adenine dinucleotide.

Cytosolic adenylate kinase (AK1) was eluted with a high yield from an affinity column of blue dextran-Sepharose 4B which bound AK1 with NADH at a low concentration but not with NAD+ at the same concentration. The difference spectrum of AK1-NADH complex against free AK1 and NADH showed positive maxima at 269nm and 273nm and a negative maximum around 326nm, and that of AK1-NAD+ complex also showed a positive maximum at 275nm. NADH and NAD+ competitively inhibited AK1 with respect to both AMP and ATP. From these results and reference data describing on topological equivalence between substrate-binding sites of AK1 and NAD-binding site of dehydrogenases, we can speculate that NAD adenine and nicotinamide moieties bind to ATP and AMP-binding sites of an AK1 molecule, respectively.

Interaction of Candida pyruvate kinase with cibacron blue

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from several yeasts were absorbed to blue dextran-Sepharose 4B and eluted with 1 M KCl in high yields. While pyruvate kinase of Candida guilliermodii AHU 4101 was eluted to some extent with phosphoenopyruvate (PEP), ADP or fructose, 1,6-diphosphate (FDP) individually from blue dextran-Sepharose 4B, the combination of FDP with PEP or ADP caused the complete elution of the kinase. Cibacron Blue competitively inhibited the pyruvate kinase with respect to both PEP and ADP at a saturated level of fixed substrate in the presence FDP, an allosteric activator. Further inhibition studies with Cibacron Blue suggest that one molecule of the blue dye binds per active site of the kinase at a saturate level of PEP, but two molecules of the dye bind per active site an unsaturated level of PEP in the presence of FDP.

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase. E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R 4) whereas the second peak with sp act of 180 corresponds to R 2R 2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R′) and 57,500 (R). On the other hand peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R 4 and R 2R 2 forms of the enzyme have the same pH optimum of 7.2.

Partial Characterization of the in Vitro Activation of Inactive Pyruvate, Pi Dikinase from Darkened Maize Leaves

In vitro activation of dark-inactivated pyruvate, orthophosphate dikinase extracted from maize (Zea mays L. cv. Golden Cross Bantam T51) leaves was examined. The inactive form of the enzyme and orthophosphate behaved kinetically as substrates for the reaction, which was catalyzed by an activating factor. This factor was bound by Blue Dextran Sepharose 4B and could be eluted by KCl at a concentration of 0.5m. The molecular weight of the maize leaf activating factor was about 88,000. Cibacron Blue 3G-A, a reactive moiety of Blue Dextran, inhibited the factor competitively with respect to the concentration of the inactive dikinase with a K(i) of 4.6 micromolar. Adenosine diphosphate and pyrophosphate were also found to be competitive inhibitors of activation, with respect to the inactive dikinase, giving K(i) values of 90 and 140 micromolar, respectively. Adenosine, other nucleotide diphosphates, and dinucleotides gave little or no inhibition of activation. These results suggest the association of a nucleotide, presumably nucleotide diphosphate, with the inactive form of pyruvate, orthophosphate dikinase.

68] Phosphoprotein phosphatase from swine kidney

This chapter describes an assay method and the purification procedure for the enzyme phosphoprotein phosphatase from swine kidney. The enzyme hydrolyzes phosphoryl groups attached to specific serine residue in polypeptide substrates and shows little or no activity with other low-molecular-weight monophosphate esters. The enzyme activity can be measured by the rate of 32 P i release from 32 P-labeled enzymes or by changes in the activity of the phosphorylated forms of phosphorylase or glycogen synthase. One enzymic assay is based on the ability of phosphoprotein phosphatase to catalyze the conversion of glycogen phosphorylase to a dephosphorylated form that is not dependent on the presence of adenosine monophosphate (AMP) for activity. The other assay is based on the ability of the phosphatase to convert glycogen synthase to a dephosphorylated form dependent on the presence of glucose 6-phosphate for activity. The purification procedure involves homogenization, acid precipitation, precipitation with ammonium sulfate, chromatography on diethylaminoethyl (DEAE)-Sephadex A-50, gel filtration on Sephacryl S-200, chromatography on Blue Dextran–Sepharose 4B and hexanediamine-Sepahrose 4B, chromatography on polylysine-Sepharose 4B, and gel filtration on Sephadex G-100. The molecular weight of the enzyme estimated by gel filtration on Sephadex G-100 is 70,000. The pH optimum of the purified enzyme is 7.0; 10 mM MgCl 2 is required for maximum activity.

Purification, crystallization and properties of 6-phospho-D-gluconate dehydrogenase from Gluconobacter suboxydans.

6-Phospho-D-gluconate dehydrogenase (EC 1.1.1.44) was purified and crystallized from a cell free extract of Gluconobacter suboxydans IFO 12528. Affinity chromatography on a blue-dextran Sepharose 4B column was effective through the purification steps, and a 280-fold purification was achieved with a 56% yield. The crystalline enzyme was homogeneous, as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. Although the enzyme from G. suboxydans catalyzed the oxidative decarboxylation of 6-phospho-D-gluconate to D-ribulose-5-phosphate, it differed from enzymes derived from other sources in respect to substrate specificity and molecular properties. Unlike the others, this Gluconobacter enzyme has a requirement for similar amounts of NAD and NADP as coenzymes and the pH optimum of this protein with two coenzymes was samely 6.5, while other enzyme preparations have the optimum pH at 8.0. The molecular weight of this enzyme was 175, 000, and the enzyme was composed of four identical subunits of molecular weight 43, 000. The enzyme activity was inhibited competitively by ATP, D-fructose-1, 6-diphosphate and NAD(P)H. It was shown that the NADPH formed by the action of the enzyme was reoxidized by the old yellow enzyme of the organism.

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.

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%).

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.

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.

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.

Crystallization and characterization of NADP-dependent D-glucose dehydrogenase from Gluconobacter suboxydans.

NADP-Dependent D-glucose dehydrogenase (EC 1.1.1.47) was crystallized for the first time from cytosol fraction of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A-50 and affinity chromatography by blue-dextran Sepharose 4B. The enzyme was purified about 1, 800-fold with an overall yield of 30%. Crystalline enzyme preparation was homogeneous in disc gel electrophoresis and analytical ultracentrifugation. The enzyme was highly specific for NADP and completely inactive with NAD. NADPH yielded in D-glucose oxidation to D-glucono-δ-lactone was reoxidized to NADP by the old yellow enzyme which existed in the same cytosol fraction of the organism. Cyclic regeneration of NADP occurred smoothly in the presence of D-glucose dehydrogenase, old yellow enzyme and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney.

Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.

Purification and partial characterization of different forms of phosphofructokinase in man

Phosphofructokinase (ATP: d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phophofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P- enzyme . Muscle and heart contain only enzyme subunits with a molecular weight of 85 000. This type of subunit is predominant in brain, where it co-exists with subunits of about 80 000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80 000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).

In Vitro Stability of Nitrate Reductase from Wheat Leaves

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH(2))-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes-methylviologen-nitrate reductase and NADH-ferricyanide reductase-also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH(2)-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH(2)-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t((1/2)) [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH(2) do not donate electrons to the same site of the nitrate reductase protein.

Crystallization and properties of 5-keto-d-gluconate reductase from Gluconobacter suboxydans.

5-Keto-d-gluconate reductase (EC 1.1.1.69) was purified and crystallized for the first time from cell-free extract of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A–50, blue-dextran Sepharose 4B, followed by pH gradient chromatography on DEAE-Sephadex A–50. The enzyme was purified about 1200-fold with an overall yield of 40%. The enzyme was much stabilized against heating and storage by adding either d-gluconate or 5-keto-d-gluconate. 2-Keto-d-gluconate had no effect to stabilize the enzyme and the enzyme was confirmed to be a different entity from 2-keto-d-gluconate reductase stabilized by gluconate or 2-keto-d-gluconate but not 5-keto-d-gluconate. It was also confirmed with crystalline enzyme that 5-keto-d-gluconate reductase is considered to have a function to reduce intracellular 5-keto-d-gluconate to d-gluconate in combination with regeneration of NADP.

Crystallization and Properties of 5-Keto-d-gluconate Reductase fromGluconobacter suboxydans

5-Keto-d-gluconate reductase (EC 1.1.1.69) was purified and crystallized for the first time from cell-free extract of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A–50, blue-dextran Sepharose 4B, followed by pH gradient chromatography on DEAE-Sephadex A–50. The enzyme was purified about 1200-fold with an overall yield of 40%. The enzyme was much stabilized against heating and storage by adding either d-gluconate or 5-keto-d-gluconate. 2-Keto-d-gluconate had no effect to stabilize the enzyme and the enzyme was confirmed to be a different entity from 2-keto-d-gluconate reductase stabilized by gluconate or 2-keto-d-gluconate but not 5-keto-d-gluconate. It was also confirmed with crystalline enzyme that 5-keto-d-gluconate reductase is considered to have a function to reduce intracellular 5-keto-d-gluconate to d-gluconate in combination with regeneration of NADP.