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Biomedical subjects

E W Weiler

Publications and source records attributed to E W Weiler.

At least 73 records · Page 4Linked to original sources
PubMed1992

Monoclonal antibody-based enzyme immunoassay for the quantitative determination of the tropane alkaloid, scopolamine.

A monoclonal antibody (SP1-4-A2) against scopolamine was produced, characterized, and used to develop a sensitive and selective, competitive enzyme immunoassay for the quantitation of the alkaloid. The assay uses nor-scopolamine-N-beta-propionic acid coupled to alkaline phosphatase as tracer and is linear from 10 pg to 10 ng of scopolamine. As little as 10 pg of scopolamine can be quantitated in an unprocessed plant extract or in human serum after suitable dilution, corresponding to detection limits of 0.1 ng scopolamine/ml of plant extract or 0.5 ng/ml of serum. The assay is more selective for scopolamine (percent cross-reactions for hyoscyamine = 0.21%, 6-hydroxy-hyoscyamine = 0.17%) than previously reported immunoassays. The assay format was designed to minimize intra- and inter-plate variabilities which are, on an average, all below 3% (coefficients of variation). The assay reported here has been validated against an HPLC-based technique using plant samples and was shown to correlate closely (y = 0.959 x + 0.14, r = 0.982).

Antibodies, Monoclonal↗
PubMed1991

Characterization and purification of the fusicoccin-binding complex from plasma membranes of Commelina communis.

The fungal phytotoxin fusicoccin binds with high affinity to plasma membranes of the monocotyledonous plant, Commelina communis L. The sites bind the toxin with an apparent Kd of 5.2 nM and a pH optimum of 6.0. They occur at a level of approximately 6-8 pmol/mg plasma membrane protein. Photoaffinity labeling with the biologically active fusicoccin derivative 9'-nor-8'-(4-azido[3,5-3H]benzoyl) diaminoethylfusicoccin identified a polypeptide of 31.5 kDa on SDS/PAGE which was strongly labeled. A second 32.5-kDa band was also consistently labeled, although not to the same extent. The binding sites were solubilized in functional form and a purification scheme was developed based on affinity and ion-exchange procedures. The purified fraction contains two polypeptides of apparent molecular masses of 30.5 kDa and 31.6 kDa. A detailed molecular analysis of the fusicoccin-binding complex is now possible.

Affinity Labels↗
PubMed1991

Domains of yeast plasma membrane and ATPase-associated glycoprotein.

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

Cell Membrane↗
PubMed1990

Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System.

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with (45)Ca(2+) through the action of the plasma membrane Ca(2+)-ATPase. While the Ca(2+)-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC(50)] = 70 micromolar) and W-7 (EC(50) = 100 micromolar), but to a much lesser extent, W-5 (EC(50) = 500 micromolar) led to a rapid efflux of (45)Ca(2+) from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La(3+), but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in (45)Ca(2+) in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca(2+)-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca(2+) from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC(50)-values of these compounds for mobilizing membrane-associated Ca(2+) (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca(2+) release from Ca(2+)-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca(2+) channel in the plasma membrane of C. communis. The channel is obtained in a Ca(2+)-inactivated state after preparation and Ca(2+)-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca(2+)-mobilizing effect of these compounds. The activated Ca(2+) channel is La(3+) sensitive and, in the cell, would allow for passage of Ca(2+) into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca(2+) influx, displaying channel properties, in a higher plant.

Journal Article↗
PubMed1990

Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L.

The protein(s) that constitute(s) the ATP-driven Ca(2+)-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca(2+)-transport system was studied using ATP-driven (45)Ca(2+) import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K(m) for Ca(2+) (5.2 micromolar), inhibition by relatively high levels of vanadate (IC(50) = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC(50) = 0.01 micromolar) and had a low apparent K(m) for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca(2+)-transport detectably in the reconstituted system. However, low levels of the Ca(2+)-ionophore A 23187 instantaneously discharged 90% of the Ca(2+) associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca(2+)-transport in the reconstituted system was thus primary active, through a Ca(2+)-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca(2+)-ATPase and for studying structural and functional aspects of the purified enzyme.

Journal Article↗
PubMed1989

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. : Characterization, Solubilization, and Photoaffinity Labeling.

Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

Journal Article↗
PubMed1988

Identification of GTP-binding proteins in the plasma membrane of higher plants.

Antisera raised against a highly conserved amino acid sequence (G alpha-common peptide) of animal Gs alpha, Gi alpha, Go alpha and Gt alpha recognize, in plasma membranes of several higher plants, sets of proteins of Mr = 37 and 31 kDa (Vicia faba), 36 and 31 kDa (Arabidopsis thaliana) and 38 and 34 kDa (Commelina communis). The A. thaliana proteins were solubilized and partially purified. They bind [35S]GTP gamma S with high affinity (apparent Kd approximately 10 nM) and, with lower affinity, GTP but not the other nucleotides tested (ATP, CTP, ITP, UTP).

Blotting, Western↗
PubMed1988

Water-stress-induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme-amplified immunoassay.

A highly sensitive, solid-phase, enzyme-amplified immunoassay for the plant growth regulator (+)-abscisic acid (ABA) was developed. The assay sensitivity (0.2-10 fmol) was sufficient for analyzing free ABA in homogeneous tissue samples dissected from Vicia faba L. leaves. Eight hours after detached leaves had been desiccated to 10% loss of fresh weight, the bulk leaf ABA content increased from </=0.2 to 6.2 ng.(mg dry weight)(-1). Epidermal tissue, spongy parenchyma cells, and palisade parenchyma cells from this water-stressed leaf had the following ABA contents, respectively: 4.8, 9.4, and 9.0 ng.(mg dry weight)(-1). Guard cells, which respond to exogenous ABA by losing solutes and volume, were also assayed. When they were dissected from control (fully hydrated) leaves, their ABA content was approximately 0.7 fg.(cell pair)(-1) [[unk]0.2 ng.(mg dry weight)(-1)]. In contrast, the ABA content of guard cells of water-stressed leaves was approximately 17.7 fg.(cell pair)(-1). These results indicate that ABA accumulation in a highly stressed V. faba leaflet is generalized; guard cells contain only 0.15% of bulk leaf ABA. The time course for loss of ABA from guard cells of a floating epidermal peel was studied. There was little loss within 30 min, but after 4 hr, the ABA content was only 17% of the original value. These results indicate that the bulk of guard cell ABA is not readily diffusible (i.e., probably not apoplastic). The results also indicate that common laboratory procedures results in lowered guard cell ABA content.

Journal Article↗
PubMed1987

Monoclonal antibodies against fusicoccin with binding characteristics similar to the putative fusicoccin receptor of higher plants.

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [(3)H]-nor-fusicoccin-alcohol of high specific activity (1.5 x 10(14)Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 x 10(-9) molar and 1.85 x 10(-9) molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 x 10(-9) molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [(3)H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe.

Journal Article↗
PubMed1986

Monoclonal Antibodies to Plant Growth Regulators: III. Zeatinriboside and Dihydrozeatinriboside.

Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed.

Journal Article↗
PubMed1985

Naringin levels in citrus tissues : I. Comparison of different antibodies and tracers for the radioimmunossay of naringin.

The preparation of a tritiated radiotracer that was used in the radioimmunoassay of naringin (naringenin-7-O-alpha-rhamnosyl- (1-2)-beta-d-glucopyranoside) and which was synthesized by reduction of the carbonyl group of the flavanone is reported. The resulting assay has a detection limit of 0.5 picomole per 0.1 milliliter, is specific for the 7-neohesperidoside substitution on flavanones, and can measure naringin in crude extracts of plant tissues. This radioimmunoassay is compared with three other naringin immunoassays which use antibodies raised against two different haptens and different tracers labeled with (125)I or (3)H. The applicability of the methods to the quantification of naringin and other flavanone neohesperidosides in citrus tissue is discussed.

Journal Article↗
PubMed1984

The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid.

Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin-controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000. We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis. Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole-3-acetamide into a substance which was unambiguously identified as indole-3-acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non-transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole-3-acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole-3-acetic acid.

Amidohydrolases↗
PubMed1984

Competitive solid phase enzyme-linked immunoassay for the quantification of limonin in citrus.

A solid-phase enzyme immunoassay for the quantitative determination of the bitter triterpene-lactone, limonin, in citrus juice samples is described. As little as 0.1 ppm of limonin can be detected. Quantitative results are available within 1 h of total assay time. The assay makes use of a limonin-alkaline phosphatase tracer of high immunoreactivity and has been semiautomated using antibody-coated polystyrene microcuvettes, a vertical light path photometer, and a forced-air microplate incubator.

Alkaline Phosphatase↗
PubMed1980

Direct radioimmunoassay for the determination of 16-acetyl-gitoxin in serum.

A simple and reliable radioimmunoassay procedure for the specific determination of 16-acetyl-gitoxin, the main cardioactive metabolite of penta-acetyl-gitoxin, in serum is described. Antisera raised against 15-acetyl-gitoxin-bovine serum albumin conjugates proved to be highly specific, thus permitting direct analysis of serum. The assay is capable of detecting 0.4 ng of 16-acetyl-gitoxin. This covers the therapeutic and toxic range when 0.1 ml serum is analyzed. A single worker can assay about 100 serum samples per day in triplicate.

Acetyldigoxins↗
PubMed1979

Iodinated digoxin derivatives with improved reactivity and stability, for use in radioimmunoassay.

We report the synthesis and properties of a new digoxin derivative, prepared by reacting cyanogen bromide-activated digoxin with tyramine. The derivative is extremely stable, and its monoiodinated form can be isolated in 85--90% purity by a single thin-layer chromatographic step. [125I]Tyramine-digoxin is immunologically stable for at least four months (immunoreactivities, 90% or greater throughout), with a shelf life of about six months. Nonspecific binding of less than 10% was observed for several batches during this time.

Digoxin↗