NADH:hydroxypyruvate reductase and NADPH:glyoxylate reductase in algae: partial purification and characterization from Chlamydomonas reinhardtii

Hydroxypyruvate and glyoxylate reductase activities were measured in extracts from the unicellular green algae, Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella miniata, and Dunaliella tertiolecta. Only trace levels of these activities were detectable in the blue-green algae, Anabaena variabilis and Synechococcus leopoliensis. A NADH-dependent hydroxypyruvate reductase was purified 130-fold from Chlamydomonas to a specific activity of 18 mumol NADH oxidized X min-1 X mg protein-1. The pH optimum was 5.0 to 7.0 in the presence of phosphate and the Km(hydroxypyruvate) was 0.05 mM. Substrate inhibition by hydroxypyruvate could be partially relieved by phosphate. The molecular weight, estimated by gel filtration, was 96,000. NADH-dependent glyoxylate reductase activity copurified with the hydroxypyruvate reductase. The Km(glyoxylate) was 10 mM, and the pH optimum was 4.5 to 8.5. A specific NADPH:glyoxylate reductase was also partially purified which did not reduce hydroxypyruvate or pyruvate. The NADPH:glyoxylate reductase had a Km(glyoxylate) of 0.1 mM and a pH optimum of 5.0 to 9.5. These reductases were compared with the pyruvate reductase of Chlamydomonas which also catalyzes the reduction of both hydroxypyruvate and glyoxylate.     

Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A

1. Phosphofructokinase from baker's yeast is partitioned between the phases of an aqueous two-phase system, containing dextran (Mr = 500000) and poly(ethyleneglycol) (Mr = 6000), in favour of the dextran-rich phase. By covalent binding of the dye Cibacron blue F3G-A to poly(ethyleneglycol) the enzyme can be extracted to the phase rich in this polymer, i.e. affinity partitioning. 2. The affinity partitioning effect, measured as the logarithmic increase of the partition coefficient by introducing polymer-bound Cibacron blue, depends on several factors. The influence of dye-polymer concentration, polymer concentration, polymer molecular weight, kind of salt and salt concentration, pH and temperature has been studied. 3. The effect of ATP, ADP, AMP, ITP, fructose 1,6-bis-phosphate and fructose 6-phosphate show large differences in the binding strength of these substances to the Cibacron blue binding sites. AMP cannot compete with Cibacron blue while ATP is strongly competing. 4. The use of affinity partitioning for enzyme isolation and determination of ligand binding is discussed, as well as possible mechanisms concerning this type of liquid/liquid extraction.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Affinity partitioning with polymer-bound Cibacron blue F3G-A for rapid,large-scale purification of phosphofructokinase from baker's yeast

Phosphofructokinase has been isolated in homogenous form from baker's yeast. The first two steps, fractional precipitation with polyethylene glycol and affinity partitioning in aqueous biphasic systems containing Cibacron blue F3G-A-polyethylene glycol, gave a 58-fold purification within 3 h. In these steps the amount of contaminating proteases was reduced by 2 orders of magnitude. After concentration using DEAE-cellulose followed by gel chromatography, homogeneous enzyme was obtained. The advantages of affinity partitioning for large-scale preparations are discussed.     

Pig heart nucleosidediphosphate kinase. Phosphorylation and interaction with Cibacron blue 3GA

The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 +/- 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron blue 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 microM) and uncompetitive with respect to 8-bromoinosine 5'-diphosphate, the acceptor nucleotide (apparent Ki = 0.31 microM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

Nitrate reductase from yeast: cultivation,partial purification and characterization

Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K _m determinations with NADH/NADPH were both 4 × 10^–4 mol/l; values for the substrate inhibition constant (K _i) were 6 × 10^–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa. Offprint requests to: R. Gromes     

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

Soluble NADH-cytochrome b5 reductase from rabbit liver cytosol: partial purification and characterization.

A soluble form of NADH-cytochrome b5 reductase (NADH: ferricytochrome b5 oxidoreductase, EC 1.6.2.2) was found in the cytosolic fraction of rabbit liver. The partially purified enzyme was strictly specific for NADH. It catalyzed the reduction of several substrates such as the methemoglobin-ferrocyanide complex (Hegesh, E. and Avron, M. (1967) Biochim. Biophys. Acta 146, 91-101) (apparent Km: 8 micrometer), potassium ferricyanide (apparent Km: 10 micrometer) and ferricytochrome b5 (apparent Km: 15 micrometer). Upon acrylamide gel isoelectro-focusing followed by specific staining, the enzyme was resolved into four bands (isoelectric pH: 7.05, 6.70, 6.50 and 6.30). The optimum pH of activity with ferricytochrome b5 as a substrate was 6.5. The estimated molecular weight was 25 000--30 000. The enzyme was unsensitive to cyanide. It was strongly inhibited by p-hydroxymercuribenzoate. The cytosolic liver cytochrome b5 reductase was immunologically related to the soluble cytochrome b5 reductase from human and rabbit red-cells, and to the microsomal cytochrome b5 reductase from rabbit liver.     

Use of Cibacron blue F 3GA-CL-sepharose 6B for the purification of T4 DNA- and RNA-ligases

The sorption capacity of the dye cibacron blue F3GA, immobilized on CL-Sepharose 6B and other support matrices, in respect to DNA- and RNA-ligases T4 was being studies. Cibacron blue F3GA immobilized on CL-Sepharose 6B binds a three-fold amount of DNA-ligase in comparison to RNA-ligase. The enzyme chromatography on cibbacron blue F3GA-CL-Sepharose 6B revealed a stronger linkage between DNA-ligase T4 and the sorbent than between RNA-ligase T4 and the sorbent. Elution was performed with potassium chloride. DNA-ligase T4 was eluted with 0.25-0.5 M KCl and RNA-ligase T4 with 0.08-0.18 M KCl. Since deoxyexonuclease contaminants possess stronger bonds with the sorbent than ligases, elution of deoxyexonucleases occurs at higher concentrations of KCl. Chromatography of enzymes on cibacron blue F3GA-CL-Sepharose 6B allows one to obtain DNA- and RNA-ligases essentially free of DNase and RNase contaminants.     

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.     

Preparation and characterisation of Cibacron Blue F3G-A poly(ethylene) hollow-fibre affinity membranes

Poly(ethylene) hollow-fibre membranes with immobilised Cibacron Blue F3G-A were obtained in four different ways from epoxy-activated fibres. Membranes with a maximum capacity of 26 mg lysozyme ml^–1and a dye density of 52 mol ml^–1were obtained when ammonia was used to open the epoxy group before dye immobilisation. Pure water flux of the modified membranes at 1 bar pressure was 1.0 cm min^–1, thus meaning only a reduction of 1.5-fold with regard to the unmodified membranes. The support-dye bond was stable as judged by the unmodified capacity of the membranes and the negligible amount of dye leaked after 520 h of exposure to 6 M urea in 0.5 M NaOH.     

Preparation and characterization of native poly(3-hydroxybutyrate) microspheres from Ralstonia eutropha

An efficient process for the preparation of poly(3-hydroxybutyrate) (PHB) microspheres with a narrow size distribution was developed. PHB was produced by a fed-batch culture of Ralstonia eutropha using fructose syrup as the sole carbon source. After autoclaving the bacteria, PHB granules, which accumulated in the cells, were isolated by a detergent/hypochlorite treatment and then spray-dried to obtain the microspheres. The diameters of the PHB microspheres ranged from 0.6 to 1.1 m and the weight-average molecular weights were approximately 50000 with polydispersity indexes of 5.0. The microspheres had a porous internal structure with an average porosity value of 72% and efficiently blocked UV light shorter than 220 nm. When isosorbide dinitrate was used as a model drug, the optimal drug loading concentration of the microspheres for controllable retardation was 3% (w/w). Almost 80% of the loaded drug (3%, w/w) was released within 12 h with typical sustained drug release behaviors.     

Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain

The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.     

Studies of the structure--function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran--sepharose and Cibacron blue 3G-A

Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvte kinase--Cibacron blue complex.     

Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase.

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.     

Dye-Ligand Affinity Chromatography: The Interaction of Cibacron Blue F3GA® with Proteins and Enzyme

Abstract

The dye Cibacron Blue F3GA has a high affinity for many proteins and enzymes. It has therefore been attached to various solid supports such as Sephadex, Sepharose, polyacrylamide, and the like. In the immobilized form the dye has rapidly been exploited as an affinity chromatographic medium to separate and purify a variety of proteins including dehydrogenases, kinases, serum albumin, interferons, several plasma proteins, and a host of other proteins. Such a diversity shown by the blue dye in binding several unrelated classes of proteins has generated considerable work in terms of studies of the chromophore itself and also the immobilized ligand. As a prelude to realizing the full potential of the immobilized Cibacron Blue F3GA, an understanding of the basic interactions of the dye with its surroundings must be gained. It has been recognized that the dye is capable of hydrophobic and/or electrostatic interactions at the instance of the ambient conditions. The study of interactions of the dye with salts, solvents, and other small molecules indicates the nature of the interactions of the dye with different kinds of groups at the interacting sites of proteins. The review will cover such interactions of the dye with the proteins, the interactions of the proteins with the immobilized ligand, and the media used to elute the bound protein in several cases, and thus consolidate the available information on such studies into a cogent and comprehensive explanation.     

Purification and characterization of hydroxypyruvate reductase from the facultative methylotroph Methylobacterium extorquens AM1.

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.     

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.