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Query: UNIPROT:P06889 (Mol)
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We have developed a method for use in investigating factors controlling the binding and cross-linking by bivalent haptens of immunoglobulin E (IgE) bound to receptors on rat basophilic leukemia (RBL) cells. This method employs monoclonal anti-2,4-dinitrophenyl (DNP) IgE that is labeled with fluorescein-5-isothiocyanate (FITC), and it measures FITC quenching that accompanies DNP occupation of the antibody combining sites in a titration experiment. The validity of this approach is demonstrated using the monovalent hapten DNP-L-lysine. The affinity constant for this ligand obtained by the FITC quenching method is compared with those obtained with previously established methods: equilibrium dialysis and quenching of endogenous tryptophan for IgE in solution and [3H]-DNP-L-lysine binding to IgE on cells. The FITC quenching method has been used to carry out a detailed study of the binding of monovalent DNP-aminocapryol-L-tyrosine (DCT) and bivalent (DCT)2-cystine to FITC-IgE and its Fab fragments in solution. Intrinsic (K) and cross-linking (Kx) affinity constants are obtained by analyzing the binding curves in terms of simple equilibrium equations. With these DCT haptens the ability of this method to assess hapten binding and cross-linking of IgE bound to receptors on RBL cells is shown.
Mol Immunol 1986 Jul
PMID:Cross-linking of IgE-receptor complexes at the cell surface: a fluorescence method for studying the binding of monovalent and bivalent haptens to IgE. 294 10

The ability of a series of bivalent haptens to bind and cross-link immunoglobulin E (IgE) in solution and on the surface of cells was examined. Several short (less than 30 A) dinitrophenyl (DNP) haptens were found to bind tightly to and cross-link a monoclonal anti-DNP IgE in solution, but these failed to trigger substantial release of 3H-serotonin from sensitized rat basophilic leukemia (RBL) cells or rat peritoneal mast cells. A longer bivalent hapten, approximately 50 A in length, consisting of two DNP-aminocaproyl-L-tyrosine (DCT) groups coupled to the alpha-amino groups of L-cystine was synthesized and characterized. This bivalent hapten [(DCT)2-cystine], binds very tightly to the same monoclonal anti-DNP IgE in solution and cross-links these antibodies to form higher mol. wt aggregates as judged by gel filtration and binding studies. It also stimulates degranulation of both RBL and mast cells sensitized with two different monoclonal anti-DNP IgE antibodies, with the mast cells exhibiting generally greater responsiveness to this ligand. The (DCT)2-cystine bivalent hapten appears to have the structural features necessary for carrying out detailed binding studies with receptor-bound IgE on the cell surface.
Mol Immunol 1986 Jul
PMID:Cross-linking of IgE-receptor complexes at the cell surface: synthesis and characterization of a long bivalent hapten that is capable of triggering mast cells and rat basophilic leukemia cells. 294 11

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.
 ...
PMID:The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors. 296 Mar 85

L-Tryptophan (4 mM) did not affect insulin release at 3 mM glucose but strongly potentiated glucose-induced (10 mM) insulin release in microdissected ob/ob mouse islets. The effect was concentration dependent with half-maximum at about 5 mM. 10 mM L-glutamate also enhanced the effect of 10 mM D-glucose on insulin release but L-phenylalanine, L-tyrosine, L-alanine, glycine and L-glutamine did not. 0.1 mM benserazide and 0.1 mM alpha-monofluoromethyldopa did not inhibit the effect of L-tryptophan. 1 mM aminooxyacetate reduced the potentiating effect of L-tryptophan but not that of L-5-hydroxytryptophan. 10 mM indole pyruvate stimulated basal insulin release but inhibited the effect of glucose. 10 mM L-glutamine did not enhance the stimulatory effect of indole pyruvate. 10 mM L-5-hydroxytryptophan reduced the effect of 10 mM L-glutamine on glucose oxidation. L-5-Hydroxytryptophan did not influence 14CO2 production from islets preloaded with [14C]glutamine but reduced the oxidation rate when [14C]glutamine was present in the incubation medium. Both L-tryptophan and L-5-hydroxytryptophan potentiate insulin release. The underlying mechanisms probably differ but do not seem to involve transaminations. The effect of L-5-hydroxytryptophan may be coupled to the activity of aromatic L-amino acid decarboxylase.
Mol Cell Endocrinol 1986 Dec
PMID:Aromatic amino acids and pancreatic islet function: a comparison of L-tryptophan and L-5-hydroxytryptophan. 354 29

An expression for the time-dependent concn of antibody in a hemispherical antigen-coated well is derived by taking the Laplace transformation of the diffusion equation. From this expression, and from antibody adsorption kinetics measured using ELISA, it is possible to evaluate the rate constant of bimolecular association, k1, the rate constant of first order antibody-antigen dissociation, k2, and their ratio, the binding equilibrium constant or affinity, Ka. For the interaction of an anti-arsanilate monoclonal antibody with arsanilate-coupled albumin, analysis yields k1 = 8.8 X 10(3) M-1 sec-1, k2 = 2.5 X 10(-4) sec-1 and Ka = 3.5 X 10(7) M-1, for mean values over 10 experiments. These results are discussed in reference to the conventionally-obtained values for binding constants, including the affinity of the anti-arsanilate monoclonal for the hapten (p-azobenzenearsonate)-N-acetyl-L-tyrosine, determined by equilibrium dialysis.
Mol Immunol 1985 Mar
PMID:ELISA-based determination of immunological binding constants. 400 Jan 35

The action of monovalent cations Li+, Na+, K+, Rb+, Cs+, NH4+ on catalytic and physico-chemical properties of bacterial tyrosine--phenol-lyase was investigated. It was shown that K+, Rb+, Cs+, NH4+ were the noncompetitive activators of the enzyme, Na+ was an inhibitor, Li+ did not influence the catalytic activity. The values of KA and Vmax were determined for the activators in the reaction of alpha, beta-elimination of L-tyrosine. Monovalent cations affect the absorption and CD spectra of the enzyme and its complex with the quasi-substrate--L-alanine. It was suggested that an activation of tyrosine phenollyase by monovalent cations was connected with the increase of the active protonated form of the holoenzyme (lambda max 420 mm) induced by the cations-activators.
Mol Biol (Mosk)
PMID:[Effect of monovalent cations on the catalytic and spectral properties of tyrosine-phenol-lyase from Citrobacter intermedius]. 403 40

The steady-state kinetics of tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] frequently exhibits complex features which confound interpretation of the results. Using an assay-enzyme system which is essentially devoid of the major mitigating kinetic features, a comprehensive kinetics data base has been compiled. The studies employed L-tyrosine, 5,6,7,8-tetrahydrobiopterin, and oxygen as substrates, and 3-(3',4'-dihydroxyphenyl)L-alanine, a deazapterin, 3-iodo-L-tyrosine, and dopamine as product, substrate analogue, and product analogue inhibitors, respectively. All three reactants were varied pairwise, and all inhibitors (except dopamine) were tested with each of the three substrates as variable substrate. The entire data base was interpreted exclusively in terms of models for classic saturation kinetics of enzyme catalysis, providing an internally consistent kinetic model and evidence for a sequential mechanism with partially ordered sequences for substrate addition and product release. Some possible mechanisms and experimental variables relating these results to more complex kinetics of tyrosine hydroxylase are considered briefly.
Mol Pharmacol 1983 Jan
PMID:Steady-state kinetics of bovine striatal tyrosine hydroxylase. 613 41

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.
J Mol Evol
PMID:Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation. 615 89

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.
Mol Cell Biol 1981 May
PMID:The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis. 615 55

Bifunctional antigens composed of one L-tyrosine-p-azobenzenearsonate (Tyr-ABA) carrier epitope and one dinitrophenyl (DNP) haptenic epitope separated by 6-aminocaproyl or polyprolyl spacers induced weak IgM anti-DNP plaque-forming cell (PFC) responses in the spleens of mice immunized intraperitoneally, without detectable IgG PFC. However, the same antigens introduced into the footpads induced IgG PFC responses in the draining lymph nodes which rose to levels greater than 100/10(6) viable lymphocytes. Moreover, the response in the lymph nodes to booster injections of antigen was characteristic of secondary T-dependent antibody responses, whereas the splenic secondary response simply mirrored the primary. The magnitude of the IgG PFC response was influenced by the size of the spacer and by the strain of mice, although genetic control did not map to the major histocompatibility complex. Prior i.p. immunization suppressed the IgG response to subsequent immunization in the footpads. This suppression could be transferred to normal syngeneic recipient mice with spleen cells from suppressed donors. Suppressor activity was eliminated by treating the spleen cells with anti-Thy-1 antibody prior to transfer, establishing the T-cell dependency of suppression. Suppression was also induced by Tyr-ABA itself, but not by DNP-lysine, indicating the epitope specificity of the suppressor cells. Thus, bifunctional antigens induce dominant suppression in the spleen but significant help in lymph nodes.
Mol Immunol 1984 Jun
PMID:Differential induction of help and suppression in mice by bifunctional antigens administered via different routes. 620 50


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