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.     

Interaction of elongation factor 2 from wheat germ with guanosine nucleotides and ribosomes.

1. The amino acid composition of wheat germ EF2 differs to some extent from that of elongation factors from mammals and bacteria. 2. The purified wheat germ EF2, similarly as the factors from other sources, is active in the: EF1-dependent polymerization of phenylalanine; ribosome-dependent GTP hydrolysis; binding of guanosine nucleotides; and ADP-ribosylation in the presence of diphtheria toxin. Fusidic acid at a concentration of 1 mM inhibits all these EF2-dependent reactions. 3. Diphtheria toxin in the presence of NAD+ inhibits polymerization of phenylalanine but does not effect GTP binding to EF2. 4. Binding of GDP to wheat germ EF2 is inhibited by ribosomes. During interaction with ribosomes, GTP in EF2-GTP complex is rapidly hydrolysed to GDP. Both GTP and 5'-guanylmethylenediphosphonate competitively inhibit formation of the ribosome-EF2-GDP complex due to the replacement of GDP from the complex. The latter is stabilized by fusidic acid.     

The NAD domain and the predicted structure for aldolase: a word of caution.

The proposal of E. Stellwagen [(1976) J. Mol Biol., 106, 903–911] that the structure of a protein can be predicted by sequence analysis provided that the protein specifically binds Cibacron blue F3GA, is not sound at least for muscle fructose bisphosphate aldolase. Contrary to the predictions we have shown that Cibacron blue does not interact directly with lysine 227 at the catalytic sites but with different sites which bind also ATP and fructose bisphosphate. We have shown also that aldolase binds 3.5 molecules of dye per subunit (dissociation constant 1.9 μm), too great a number to support the hypothesis that the binding of Cibacron blue is a specific indication of the presence of an NAD domain.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

Effect of cibacron blue on tubulin assembly in the absence and presence of microtubule-associated proteins

Cibacron blue was found to inhibit assembly and increase the critical concentration of microtubule proteins. In the presence of 4 mol Cibacron blue/mol tubulin, assembly was completely inhibited and pre-formed microtubules disassembled. Addition of 8% (v/v) dimethylsulfoxide to Cibacron blue-inhibited samples induced assembly of normal microtubules in addition to sheets of protofilaments. Disassembly was induced upon addition of 1 mM colchicine or 2mM Ca²⁺. No obvious difference was seen in the protein composition of these microtubules compared with controls. GTP exchange was not affected by the presence of Cibacron blue nor was GTP able to counteract its effect. This indicates that the exchangeable GTP site is not involved. The extent of assembly of phosphocellulose purified tubulin in the presence of 8% (v/v) dimethylsulfoxide was only slightly less in the presence of Cibacron blue, although the assembly rate was decreased. These results suggest that Cibacron blue might alter the binding of one or more of the associated proteins stimulating assembly.     

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (K_a) and stoichiometry (r) for the enzyme-dye complex.     

Human serum albumin chromatography by Cibacron Blue F3GA-derived microporous polyamide hollow-fiber affinity membranes

An affinity dye ligand, Cibacron Blue F3GA was covalently attached onto commercially available microporous polyamide hollow-fibre membranes for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Cibacron Blue F3GA were incorporated on the polyamide hollow-fibres by changing the dye attachment conditions, i.e. initial dye concentration, addition of sodium carbonate and sodium chloride. The maximum amount of Cibacron Blue F3GA attachment was obtained at 42.5 μmol g⁻¹ when the hollow-fibres were treated with 3 M HCl for 30 min before performing the dye attachment. HSA adsorption onto unmodified and Cibacron Blue F3GA-derived polyamide hollow-fibre membranes was investigated batchwise. The non-specific adsorption of HSA was very low (6.0 mg g⁻¹ hollow-fibre). Cibacron Blue F3GA attachment onto the hollow-fibres significantly increased the HSA adsorption (147 mg g⁻¹ hollow-fibre). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (230 mg HSA g⁻¹ hollow-fibre). Desorption of HSA from Cibacron Blue F3GA derived hollow-fibres was obtained using 0.1 M Tris–HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cibacron Blue F3GA derived polyamide hollow-fibre without significant decreases in the adsorption capacities.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2±0.2 M and 11.2±0.2 M at 5 °C and 20°C respectively.This revised version was published online in October 2005 with corrections to the Cover Date.     

Distinction between cofactor-dependent and-independent phosphoglycerate mutases by chromatography on Cibacron Blue-Sepharose

The binding of phosphoglycerate mutases from a variety of sources to Cibacron Blue-Sepharose has been examined. Those enzymes which are dependent on 2,3-bisphosphoglycerate (BPG) for activity bind to the immobilized dye and can be eluted by BPG. Those enzymes which are independent of BPG do not bind to the immobilized dye. The possible structural signifi-cance of this distinction is discussed.     

The anticalmodulin effect of aflatoxin B1 on purified erythrocyte Ca2+-AtPase

The genotoxic carcinogen aflatoxin B₁ (AFB₁) inhibited the calmodulin-stimulated membrane-bound (Ca²⁺Mg²⁺)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB₁ caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca²⁺-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca²⁺-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB₁ did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB₁ inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB₁ inhibits the activated Ca²⁺-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca²⁺-calmodulin or acidic phospholipids. The implication of these observations is that Ca²⁺-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB₁ because of its anticalmodulin effect.Abbreviations ATP adenosine 5-triphosphate - EGTA ethylenglycolbis (-aminoethylether) N,N-tetraacetic acid - Hepes 4-(2 hydroxyethyl)-1-piperazine ethanesulphonic acid - AFB₁ aflatoxin B₁ - PMSF phenylmethylsulfonylfluoride - TLCK N--p-tosyl-L-lysine chloromethyl ketone - PC phosphatidycholine - PS phosphatidylserine - PI phosphatidyl inositol - DPG diphosphatidyl glycerol - SDS sodium dodecyl sulphate - Tris-HCl Tris (hydroxymethyl)aminomethane hydrochloride     

Anthrax toxins

Bacillus anthracis, the etiological agent of anthrax, secretes three polypeptides that assemble into toxic complexes on the cell surfaces of the host it infects. One of these polypeptides, protective antigen (PA), binds to the integrin-like domains of ubiquitously expressed membrane proteins of mammalian cells. PA is then cleaved by membrane endoproteases of the furin family. Cleaved PA molecules assemble into heptamers, which can then associate with the two other secreted polypeptides: edema factor (EF) and/or lethal factor (LF). The heptamers of PA are relocalized to lipid rafts where they are quickly endocytosed and routed to an acidic compartment. The low pH triggers a conformational change in the heptamers, resulting in the formation of cation-specific channels and the translocation of EF/LF. EF is a calcium- and calmodulin-dependent adenylate cyclase that dramatically raises the intracellular concentration of cyclic adenosine monophosphate (cAMP). LF is a zinc-dependent endoprotease that cleaves the amino terminus of mitogen-activated protein kinase kinases (Meks). Cleaved Meks cannot bind to their substrates and have reduced kinase activity, resulting in alterations of the signaling pathways they govern. The structures of PA, PA heptamer, EF, and LF have been solved and much is now known about the molecular details of the intoxication mechanism. The in vivo action of the toxins, on the other hand, is still poorly understood and hotly debated. A better understanding of the toxins will help in the design of much-needed anti-toxin drugs and the development of new toxin-based medical applications.Abbreviations CMG2 Capillary morphogenesis protein 2 - DTA Diphtheria toxin A chain - EF Edema factor - EFn N-terminal fragment of EF - ETx Edema toxin - GR Glucocorticoid receptors - GSK3 Glycogen synthase kinase 3 - I domain Integrin-like domain - iNOS Inducible nitric oxide synthase - LF Lethal factor - LFn N-terminal fragment of LF - LTx Lethal toxin - MAPK Mitogen-activated protein kinase - Mek MAPK kinases - PA Protective antigen - PA₂₀ 20-kDa N-terminal fragment of PA - PA₆₃ 63-kDa C-terminal fragment of PA - TEM8 Tumor endothelial marker 8     

Studies on the binding of bacterial elongation factors EF Tu and EF G to ribosomes

When EF G² from Escherichia coli or Pseudomonas fluorescens is pre-bound to ribosomes in the presence of GMD, or GTP and fusidic acid, a differential effect is observed on the subsequent EF Tu-catalyzed binding of aminoacyl-tRNA to ribosomes. The EF G from E. coli nearly completely prevents the binding reaction, whereas the corresponding factor from P. fluorescens displays a significantly lower inhibitory effect. Both EF G factors form stable complexes with ribosomes and are equally efficient in the polymerization reaction. The difference in inhibitory properties between the two factors persists over a wide range of NH₄Cl concentration.     

Dinucleotide fold proteins. Interaction of arabinose binding protein with Cibacron Blue 3G-A.

Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L-arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier (Thompson et al., 1975; Stellwagen, 1977). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.     

Hybridization of tRNAs of Drosophila melanogaster to the region of the 5S RNA genes of the polytene chromosomes

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled ¹²⁵I-tRNA^Asp ₂ occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNA^Asp ₂ labeled by introducing ¹²⁵I-5-iodocytidylyl residues into the 3-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to its other sites. The hybridization of tRNA^Lys ₂, tRNA^Gly ₃ and tRNA^Met ₃ at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNA^Lys ₂ no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNA^Asp ₂ in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.     

A colorimetric assay for steady-state analyses of iodo- and bromoperoxidase activities

The standard assay for iodoperoxidase activity is based on the spectrophotometric detection of triiodide formed during the enzymatic reaction. However, chemical instability of has limited the method to high iodide concentrations and acidic conditions. Here we describe a simple spectrophotometric assay for the determination of iodoperoxidase activities of vanadium haloperoxidases based on the halogenation of thymol blue. The relation between color and chemical entities produced by the vHPO/H₂O₂/I⁻ catalytic system was characterized. The method was extended to bromine and, for the first time, allowed measurement of both iodo- and bromoperoxidase activities using the same assay. The kinetic parameters (K_m and k_cat) of bromide and iodide for vanadium bromoperoxidase from Ascophyllum nodosum were determined at pH 8.0 from steady-state kinetic analyses. The results are concordant with an ordered two-substrate mechanism. It is proposed that halide selectivity is guided by the chemical reactivity of peroxovanadium intermediate rather than substrate binding. This method is superior to the standard assay, and we believe that it will find applications for the characterization of other vanadium as well as heme haloperoxidases.     

Complexes of Cu with mixed-donor phenanthroline-containing macrocycles: analysis of their structural, redox and spectral properties in the context of Type-1 blue copper proteins biomimetic models

The macrocycles L¹-L³ having N₂S₂O-, N₂S₂-, and N₂S₃-donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu^II to give 1:1 [M(L)]²⁺ complex species. The compounds [Cu(L¹)(ClO₄)]ClO₄ (1), [Cu(L²)(ClO₄)]ClO₄ ·  (2) and [Cu(L³)](ClO₄)₂ (3) were isolated at the solid state and the first two also characterised by X-ray diffraction studies. The conformation adopted by L¹ and L² in the cation complexes reveals the aliphatic portion of the rings folded over the plane containing the heteroaromatic moiety with the ligands encapsulating the metal centre within their cavity by imposing, respectively, a square-based pyramidal and a square planar geometry. In both complexes, the metal ion completes its coordination sphere by interacting with a ClO₄⁻ ligand. The compound [Cu(L³)₂](PF₆)₂ (4) containing a 1:2 cation complex was also isolated at the solid state: EPR spectroscopy measurements suggest the presence of a CuN₄ chromophore in this complex. The EPR and electronic spectral features of 1-4 have been studied and their redox properties examined in comparison with those observed for Type-1 blue copper proteins.The reactivity of L¹-L³ has also been tested toward stoichiometric amounts of the Cu^I salt [CuCl(PPh₃)₃].     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

GTP-dependent dopamine reception in central nervous system tissue of the pond snail Lymnaea stagnalis

The guanosine-5-triphosphate (GTP) binding of D₁-dopamine (DA) receptor agonist [H³]-SKF 38393 is described. The binding of [H³]-SKF 38393 occurs in two different DA receptors in the presence of guanylyl nucleotides, and in one receptor population in the absence of guanylyl nucleotides. It was shown with GDP--P³³ binding analysis that G proteins in the mollusc nervous tissue membranes accelerate exchange of guanosine-5-diphosphate (GDP) for GTP considerably. While binding of [H³]-SKF 38393 was not found with phosphorylation of the membranes by the catalytic subunit of cAMP-dependent protein kinase A, basal and DA-induced GDP GTP exchange was noticeably inhibited with phosphylation in the nervous tissue membranes.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 451–461, July–August, 1992.     

Probing of Various Physiologically Relevant Metals: Amyloid-β Peptide Interactions with a Lipid Membrane-Immobilized Protein Nanopore

One of the prevailing paradigms regarding the onset of Alzheimer’s disease endows metal ions with key roles in certain steps of the amyloid-β (Aβ) peptide aggregation cascade, through peptide conformational changes induced by metal binding. Herein, we focused on the truncated, more soluble Aβ_1–16 peptide fragment from the human Aβ_1–40, and demonstrated the utility of a sensing element based on the α-hemolysin (α-HL) protein to examine and compare at single-molecule level the interactions between such peptides and various metals. By using the same approach, we quantified Cu²⁺ and Zn²⁺ binding affinities to the Aβ_1–16 fragment, whereas the statistical analysis of blockages induced by a single Aβ_1–16 peptide on the current flow through an open α-HL pore show that the metal propensity to interacting with the peptide and entailing conformational changes obey the following order: Cu²⁺ > Zn²⁺ > Fe³⁺ > Al³⁺.