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L D Ward

Publications and source records attributed to L D Ward.

At least 55 records · Page 3Linked to original sources
PubMed1991

Allergenicity and physicochemical characterization of house dust mite derived amylase.

The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds.

Allergens↗
PubMed1990

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis.

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.

Amino Acid Sequence↗
PubMed1990

Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels.

A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.

Amino Acid Sequence↗
PubMed1990

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

Amino Acid Sequence↗
PubMed1989

Stimulation of microtubule assembly by recombinant human interferon-alpha and interferon-gamma.

Interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) have been demonstrated to stimulate microtubule assembly measured in the in vitro assembly system. The process is substoichiometric occurring when the interferon concentrations are below that of tubulin. IFN-gamma is a more potent effector than IFN-alpha. The critical tubulin concentration describing microtubule assembly decreases from 1.5 mg/ml measured in the absence of added effector to 1.05 mg/ml and 1.3 mg/ml when measured in the presence of 2.16.10(-6) M IFN-gamma and 3.06.10(-6) M IFN-alpha, respectively.

Animals↗
PubMed1989

Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes. A chromatographic procedure for separating proteins from detergents.

Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.

Amino Acid Sequence↗
PubMed1988

Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Adenylyl Cyclases↗
PubMed1988

Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes.

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.

Animals↗
PubMed1988

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin.

The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1988

Synthesis and characterization of CGP-12177-NBD: a fluorescent beta-adrenergic receptor probe.

The synthesis and properties of a fluorescent derivative of the hydrophilic beta-adrenergic antagonist CGP-12177 are described. The fluorescence of the NBD derivative of CGP-12177 (CGP-NBD) is extremely sensitive to its environment, the quantum yield increasing 23-fold upon transfer from water to acetonitrile. This property of CGP-NBD was taken into account and a procedure was developed using quantitative chloroform extraction of ligand for the measurement of CGP-NBD bound specifically to beta-receptors on A431.E3 membranes. The fluorescent NBD-derivative of CGP-12177 bound strongly and specifically to A431 cells, a KD of 3.9 x 10(-10) M being measured; the specific binding represented 63% of the total binding at a concentration of 1 x 10(-8) M (256 x KD). A431.E3 cells were used for the binding studies since they gave consistently higher receptor numbers when compared with the native strain. A maximal number of 47,000 sites/cell and a KD of 100 pM were measured with CGP-12177 on adhered cells. The receptor number was strongly dependent upon cell density with only 3000 sites/cell being measured in suspension at confluence.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Self-association of sperm whale metmyoglobin.

The solution behavior of sperm whale metmyoglobin in 0.15 I phosphate-chloride buffer, pH 7.2, has been examined by sedimentation equilibrium, frontal gel chromatography, and sedimentation velocity. Results obtained from all three studies are shown to be consistent with a self-association model in which dimerization of the myoglobin is governed by an association equilibrium constant of 0.068 liter/g (580 M-1) at 20 degrees C.

Animals↗
PubMed1983

Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.

Animals↗
PubMed1983

Effects of thermodynamic nonideality in kinetic studies.

Experimental evidence is presented for concentration dependence of the pseudo-first-order rate constant describing the rate of inversion of sucrose by 2 M HCl; and also of the increase in maximal velocity for the catalytic reduction of pyruvate by lactate dehydrogenase that results from addition of the inert macromolecular solutes bovine serum albumin, ovalbumin, and Dextran T70. These somewhat unusual and seemingly diverse observations are examined in terms of a theory formulated on the basis of two equilibrium reactions, the first describing complex formation between two reactants, and the second isomerization of that complex to an activated state prior to product formation. This formulation permits consideration of activity coefficient ratios relevant to the equilibria and the expression of these ratios as power series in total solution composition. Quantitative assessment of the experimental results is made possible in these terms by estimating the magnitudes of the constant coefficients of the virial expansions as excluded volumes. It is concluded that the result observed in the sucrose inversion study finds rational explanation in thermodynamic nonideality factors governing the overall equilibrium between the reactants and the activated complex of sucrose and hydronium ion. For the enzyme-catalyzed reaction the same general equation applies but particular attention is given to the simplified form that is relevant to high substrate concentrations, where, in the absence of inert compounds, the conventional maximal velocity is approached. In this region an increase in velocity observed upon addition of an inert macromolecular component may be considered explicitly in terms of excluded volume effects related to a shape change in the isomerization between enzyme-substrate complex and its activated state.

Animals↗