Biomedical subjects

T N Wells

Publications and source records attributed to T N Wells.

At least 127 records · Page 7Linked to original sources

PubMed1993

A novel dimer configuration revealed by the crystal structure at 2.4 A resolution of human interleukin-5.

Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.

Animals↗

PubMed1993

Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.

Amino Acid Sequence↗

PubMed1993

Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin-5 from Escherichia coli.

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.

Animals↗

PubMed1993

Crystallization and preliminary X-ray diffraction studies of recombinant human interleukin-5.

Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C. The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees. They diffract to at least 2.0 A resolution on a rotating anode X-ray source. The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.

Crystallization↗

PubMed1993

Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites.

Phosphomannose isomerase (PMI) from Saccharomyces cerevisiae is a zinc-dependent metalloenzyme. Besides its role in catalysis, zinc is also a potent inhibitor of the enzyme. The inhibition is competitive with the substrate mannose 6-phosphate, with Kis = 6.4 microM in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees C. This inhibition constant is 4 orders of magnitude smaller than for group II divalent cations, indicating that the binding is not primarily electrostatic. Micromolar inhibition is also observed with ions of the other metals of the electronic configuration d10. Under identical conditions, cadmium is a predominantly competitive inhibitor with Kis = 19.5 microM. Inhibition by mercury is predominantly competitive with Kis = 6.0 microM but shows a hyperbolic Dixon plot. Theorell-Yonetani double-inhibition analysis shows that zinc and cadmium ions are mutually exclusive inhibitors against mannose 6-phosphate. However, analysis of zinc and mercury double inhibition shows that they can simultaneously bind in the mannose 6-phosphate binding pocket, with only a small mutual repulsion. Inhibition of the enzyme by cadmium and zinc ions is strongly pH dependent with pKa = 9.2 for cadmium and one pKa at 6.6 and two at 8.9 for zinc. The inhibitory species are the monohydroxide forms, Zn(OH)+ and Cd(OH)+. However, inhibition by mercury is relatively pH-independent, consistent with the neutral Hg(OH)2 being the inhibitory species. In all three cases, the metal ion binding causes a conformational change in the enzyme as judged by tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1993

Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample.

The T4 bacteriophage has been used to investigate protocols for the preparation of samples for scanning force microscopy in air, in order to obtaining reproducible images. The resolution of images and the distribution of bacteriophages on the substrate depends on the buffer type, its concentration, the surface treatment of substrate, and the method of deposition. The best imaging conditions for the phages require dilution in a volatile buffer at low ionic strength and adsorption onto hydrophilic surfaces. When imaging with the scanning force microscopy the quality of the images is influenced by the vertical and lateral forces applied on the sample and by the tip geometry.

Bacteriophage T4↗

PubMed1992

ATP-citrate lyase as a target for hypolipidemic intervention. Sulfoximine and 3-hydroxy-beta-lactam containing analogues of citric acid as potential tight-binding inhibitors.

Citric acid analogues (+/-)-12a,b and (+/-)-17a,b, where one of the primary carboxylates has been replaced by a sulfoximinoyl and a 3-(3-hydroxy-beta-lactamyl) moiety, respectively, have been synthesized and evaluated as inhibitors of ATP-citrate lyase. The design of these inhibitors was based on methionine sulfoximine and tabtoxinine beta-lactam, potent, tight-binding inhibitors of glutamine synthetase. Both ATP-citrate lyase and glutamine synthetase employ phosphate-carboxylate anhydrides as a method for carboxylate activation during catalysis. Only one diastereomer, (+/-)-12a, displayed weak, reversible inhibition, while the remaining citrate analogues (+/-)-12b and (+/-)-17a,b were inactive against the lyase. No time-dependent inactivation of the enzyme was observed.

ATP Citrate (pro-S)-Lyase↗

PubMed1992

Human interleukin-5 expressed in Escherichia coli has N-terminal modifications.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity. In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus. These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry. The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.

Chromatography, High Pressure Liquid↗

PubMed1992

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.

ATP Citrate (pro-S)-Lyase↗

PubMed1992

Redox control of catalysis in ATP-citrate lysate from rat liver.

In thiol redox buffers at pH 8.0, rat liver ATP-citrate lyase is in equilibrium between an oxidised inactive form and a reduced active form. The reduced enzyme is inactivated by oxidised glutathione (GSSG) at a rate of 45 min-1.M-1 and the oxidised enzyme is activated by reduced glutathione (GSH) at a rate of 3.2 min-1.M-1. At redox equilibrium, the enzyme activity depends on the ratio [GSH]2/[GSSG]. The inactivation involves formation of a protein-protein disulphide rather than a protein-glutathione complex. This reaction has Keq = 78 +/- 7 mM for the oxidative reaction. Activity can therefore be controlled by the redox state of the cell, being more active in the fed state than in the oxidatively stressed state. This redox process is also important in the in vitro enzyme assay, where ATP-citrate lyase is in redox equilibrium with oxygen and either dithiothreitol or 2-mercaptoethanol. Reduction is a two-step process, requiring high concentrations of reductant for full activation (30 mM dithiothreitol or 200 mM 2-mercaptoethanol). The enzyme inhibitor, Medica-16 raises the redox equilibrium constant to greater than 400 mM. It binds more tightly to the oxidised form of the enzyme, with Ki less than 40 microM compared to 180 microM for the reduced form.

ATP Citrate (pro-S)-Lyase↗

PubMed1992

Imaging of proteins by scanning tunnelling microscopy.

Scanning tunnelling microscopy has been used to examine the structure of proteins deposited on a graphite surface. Three molecules have been studied; immunoglobulin G (IgG), Complement component 1q (C1q) and ATP-citrate lyase (ACL). The images show IgG as a tri-lobed molecule, consistent with the known 3D structure as determined by X-ray crystallography. The C1q images differ from the well known "tulip bunch" model derived by electron microscopy, but are consistent with the model if it is assumed that the six globular heads have aggregated. Molecules of ACL are visible as discrete units, with some hints of substructure. These results highlight the potential of STM in studying protein structures, but also illustrate the difficulties of interpreting micrographs of proteins whose structure is currently unknown.

Complement C1q↗

PubMed1991

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.

ATP Citrate (pro-S)-Lyase↗

PubMed1991

Structure of immunoglobulin G by scanning tunnelling microscopy.

Scanning tunnelling microscopy (STM) has been used to examine the shape of individual immunoglobulin G (IgG) molecules deposited onto a graphite surface. IgG was chosen for this study as it has a well-characterized and distinctive three-dimensional structure. The micrographs clearly reveal the IgG molecule as trilobed, corresponding with the known structural organization of IgG. Comparison of these images with the structure of IgG determined by X-ray crystallography shows that the STM images are consistent with the crystal structure. This illustrates that STM is a valuable technique for examining protein structure, allowing rapid determination of the overall molecular shape that is consistent with more established techniques.

Immunoglobulin G↗

PubMed1991

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.

ATP Citrate (pro-S)-Lyase↗

PubMed1991

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.

Adenosine Triphosphate↗

PubMed1991

Linear free energy relationships in enzyme binding interactions studied by protein engineering.

Experiments on mutants of tyrosyl-tRNA synthetase have shown that there can be linear free energy relationships (LFERs) between changes in activation free energies and changes in binding energies when groups are deleted that bind to non-reacting parts of the substrate (Fersht et al., 1986, 1987). It has now been proposed (Straub and Karplus, 1990) that such LFERs can occur for the mutation of hydrogen bonding groups only for the limiting examples of Brønsted beta of 0, 1 or infinity, and that fractional values of beta are not permissible. The reasoning behind this is that the energy of a hydrogen bond is not linear with distance and the (false) premise that an LFER requires that there is a linear relationship between bond energy and distance. We show from a simple model how LFERs can arise for binding interactions and how they can give fractional values of beta, in accord with experimental evidence. An LFER occurs between binding and catalysis when a set of interactions exists in which each member contributes to the binding energy of the transition state the same fraction of the binding energy it contributes to the products (both relative to the ground state).

Binding, Competitive↗

PubMed1990

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing.

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.

Adrenal Glands↗

PubMed1990

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age.

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.

ATP Citrate (pro-S)-Lyase↗