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Biomedical subjects

I Pecht

Publications and source records attributed to I Pecht.

At least 73 records · Page 4Linked to original sources
PubMed1995

The 2,4-dinitrophenyl group for protection of hydroxyl function of tyrosine during solid-phase peptide synthesis.

The facile thiolytic cleavage of the O-2,4-dinitrophenyl (Dnp) tyrosine bound was applied to the solid-phase synthesis of the 22-amino acid residue peptide H-Asp-Ala-Val-Tyr-Thr-Gly-Leu-Asn-Thr-Arg-Asn-Gln-Glu-Thr-Tyr-Glu-Thr-Le u-Lys- His-Glu-Lys-OH, corresponding to positions 62-83 in the chain of the type 1 receptor for Fc epsilon domains expressed on the rat mucosal-type mast cells (line RBL-2H3). A method for the spectrophotometric determination of insoluble O-Dnp as well as of unprotected phenolic moieties of tyrosine was developed. It is based on monitoring S-Dnp-2-mercaptoethanol, produced upon O-Dnp thiolysis by 2-mercaptoethanol.

Amino Acid Sequence↗
PubMed1995

Blockade of capacitive Ca2+ influx by Cl- channel blockers inhibits secretion from rat mucosal-type mast cells.

Whole-cell patch-clamp recordings of membrane currents were performed in combination with measurements of mediator secretion from mucosal-type mast cells (rat basophilic leukemia cells, subline 2H3), to determine the involvement of membrane conductances induced upon depletion of intracellular Ca2+ stores. In patch-clamp experiments, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-induced depletion of internal Ca2+ stores led to activation of two distinct membrane conductances, a Ca2+ current and a Cl- current. The Ca2+ current was blocked by 100 microM La3+, which did not affect the Cl- current. In contrast, 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid produced selective blocked of the Cl- current. Remarkably, the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, and N-phenylanthranilic acid (NPAA) inhibited not only the Cl- current but also the Ca2+ current. IC50 values for the blockade of the Ca2+ inward current by NPPB, niflumic acid, and NPAA were determined to be 23, 150, and 190 microM, respectively. In secretion experiments, thapsigargin-induced depletion of internal Ca2+ stores stimulated serotonin release, which was found to be strictly dependent on extracellular Ca2+. In the presence of 100 microM La3+ secretion was almost completely inhibited. In contrast, only 50% of secretion was suppressed by 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid, which fully blocked the Cl- current without affecting Ca2+ influx, as monitored by electrophysiological experiments. The other Cl- channel blockers produced a very different pattern for the inhibitory dose dependence of secretion, with IC50 values for NPPB, niflumic acid, and NPAA of 23, 60, and 180 microM, respectively. Taken together, these findings suggest that Ca2+ store depletion leads to concomitant activation of Cl- and Ca2+ currents. Blockade of the latter is apparently an additional mode of action for diarylaminocarboxylate-type Cl- channel blockers inhibiting mast cell secretory responses.

Animals↗
PubMed1994

Intramolecular electron transfer in ascorbate oxidase is enhanced in the presence of oxygen.

Intramolecular electron transfer from the type 1 copper center to the type 3 copper(II) pair is induced in the multi-copper enzyme, ascorbate oxidase, following pulse radiolytic reduction of the type 1 Cu(II) ion. In the presence of a slight excess of dioxygen over ascorbate oxidase, interaction between the trinuclear copper center and O2 is observed even with singly reduced ascorbate oxidase molecules. Under these conditions, the rate constant for intramolecular electron transfer from type 1 Cu(I) to type 3 Cu(II) increases 5-fold to 1100 +/- 300 s-1 (20 degrees C, pH 5.8) as compared to that of the same process under anaerobic conditions. This observation constitutes evidence for control of the internal electron transfer process by one of its substrates. The structural and functional significance of these findings are discussed.

Ascorbate Oxidase↗
PubMed1994

Kinetics of Fc epsilon RI dimer formation by specific monoclonal antibodies on mast cells.

Clustering of the type I receptor for Fc epsilon domains constitutes the signal initiation leading to mast cell secretory response. In order to characterize the relationship between the lifetime of clustered Fc epsilon receptors and the cellular response we have studied the rates of association and dissociation of monoclonal, IgG class antibodies (mAbs) specific for the alpha-subunit of type 1 receptor for IgE (Fc epsilon RI) (designated as F4, J17, and H10) to and from this receptor on live rat mucosa-type mast cells (line RBL-2H3) were measured at three different temperatures (25, 15, and 4 degrees C). These antibodies dimerize the Fc epsilon RI on these cells and induce their secretion, thus providing clear evidence that Fc epsilon receptor dimers are sufficient for the stimulus [Ortega et al. (1988), EMBO J. 7, 4101]. Marked differences in the response to the different mAbs have been explained in terms of possible orientational constraints imposed by them on the Fc epsilon receptor dimers. Interaction kinetics between the Fab fragments of these mAbs and the Fc epsilon RI have previously been measured and found to be best fitted by a two-reaction-step model involving a conformational transition from a low-affinity (l) to a high-affinity (h) state of the receptor-ligand complex [Ortega et al. (1991) Biochemistry 30, 3473]. Analysis of the interaction kinetics between the corresponding intact mAbs and the Fc epsilon RI therefore requires consideration of this 1-->h transition for both complexes involved, namely, the monomeric Fc epsilon RI-mAb and the dimeric Fc epsilon RI-mAb-Fc epsilon RI complexes. This was done by assuming the involvement of the following Fc epsilon RI dimer species: all 1- or h-state dimers Dll and Dhh and a hybrid Dlh with one receptor in the l state and the other in the h state. A self-consistent set of rate constants was derived by fitting the experimental results to this model. At 25 degrees C the all-h-state dimers Dhh turned out to be preferentially stabilized, probably by interaction with other cellular components. Different dimer formation rates were observed for each of the three mAbs, indicating that the dimer distribution among different states is determined by the individual epitope-binding site combination and also by the geometry of the respective complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Protein tyrosine phosphatase activity enhancement is induced upon Fc epsilon receptor activation of mast cells.

Immunological stimulation of rat mucosal type mast cells (line RBL-2H3) by clustering the type I Fc epsilon receptor (Fc epsilon RI) causes a fast and transient tyrosine phosphorylation of several proteins. This implied the involvement of both, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) in that process. In order to identify the PTPases involved in these very early steps coupling Fc epsilon RI stimulus to cell response, we undertook the purification and characterization of PTPases present in RBL-2H3 cells. In one of the cells' membranal fractions, a PTPase activity was found to be enhanced 2- to 3-fold upon cell stimulation by Fc epsilon RI clustering. Characterization of this activity implies its involvement in control of the Fc epsilon RI signalling cascade.

Animals↗
PubMed1994

Direct binding of myelin basic protein and synthetic copolymer 1 to class II major histocompatibility complex molecules on living antigen-presenting cells--specificity and promiscuity.

Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and is being tested as a candidate drug for multiple sclerosis. It has been previously demonstrated that Cop 1 is immunologically cross-reactive with the autoantigen myelin basic protein (BP) and competitively inhibits the response to BP of T-cell lines and clones of different major histocompatibility complex (MHC) restrictions, of both mouse and human origin. In the present study we demonstrated the direct binding of Cop 1, using its biotinylated derivative, to MHC molecules on living antigen-presenting cells. Binding of biotinylated BP and peptide p84-102 (an immunodominant epitope of BP) was also demonstrated. Cop 1 and BP bound in a promiscuous manner to different types of antigen-presenting cells of various H-2 and HLA haplotypes. The specificity of the binding was confirmed by its inhibition with either the relevant anti-MHC class II antibodies or unlabeled analogs. Cop 1 exhibited the most extensive and fast binding to antigen-presenting cells. In addition, Cop 1 inhibited the binding of biotinylated derivatives of BP and of p84-102 to the MHC class II molecules and even displaced these antigens when already bound. Thus, these results suggest that Cop 1 indeed competes with BP for MHC binding and, thereby, inhibits T-cell responses to BP. The binding of Cop 1 to different DR alleles, probably because of its multiple MHC binding motifs, may indicate its potential as a broad-spectrum drug for multiple sclerosis.

Amino Acid Sequence↗
PubMed1994

Protein-tyrosine kinase activity tightly associated with human type II Fc gamma receptors.

Stimulation of B cells by clustering their surface immunoglobulins (sIg) leads to enhanced phosphorylation of several cellular proteins on Ser and Tyr residues. The type II Fc gamma receptor (Fc gamma RII) is one of those proteins that undergo Ser phosphorylation. Upon affinity isolation of the Fc gamma RII, several molecular entities are coisolated from Triton X-100 lysates of BL41 Burkitt lymphoma line which undergo "in vitro" (cell free) phosphorylation in the immune complex-associated kinase assay. Furthermore, several molecules phosphorylated on Tyr upon sIgM cross-linking in the intact cells are coisolated with Fc gamma RII. The 59-kDa coprecipitated component is identified as the protein-tyrosine kinase (PTK) fyn. Clustering the sIgM molecules enhanced the in vitro phosphorylation of all molecules coprecipitated with Fc gamma RII as well as that of the exogenously added PTK substrate, enolase. Kinase renaturation assays suggest that at least two major renaturable protein kinases (59 kDa and 85-90 kDa) associate with Fc gamma RII. Whereas the 59-kDa component comigrates with the PTK fyn, the 85- to 90-kDa one is an unidentified Ser/Thr kinase. These data suggest that Fc gamma RII exists in the B-cell membrane as part of a multimolecular complex including protein kinases, activities of which are regulated by clustering of the antigen receptors.

Animals↗
PubMed1994

Picosecond fluorescence spectroscopy of a single-chain class I major histocompatibility complex encoded protein in its peptide loaded and unloaded states.

The tryptophan fluorescence properties of two different peptide complexes of the single-chain H-2Kd (SC-Kd) were studied by means of the single-photon counting technique. The latter enables time-resolved measurements of fluorescence intensity and anisotropy decay parameters relevant to structural and dynamic properties of proteins. While the isolated SC-Kd molecules in their 'original' purified form represent the unloaded state, i.e., containing endogenous low-affinity peptides, the loaded SC-Kd protein is obtained by introducing well-defined high-affinity peptides that replace the low-affinity ones. These two SC-Kd forms were found to exhibit different time-resolved tryptophan emission patterns; the unloaded complexes show a slightly faster fluorescence intensity decay rate than the loaded one. Three well-resolved time domains were distinguished in the anisotropy decay course of both forms: a short one in the picosecond range, an intermediate one of several nanoseconds, and a long one spanning several dozens to hundreds of nanoseconds. They are assigned to superposition contributions of (short- and long-distance) non-radiative energy transfer processes, to motions of the tryptophans, and to rotation of the whole protein globule. In the loaded SC-Kds, the first two processes were found to be attenuated. It is therefore suggested that upon binding of high-affinity peptides, the SC-Kd structure becomes more compact and certain tryptophans become less accessible to quenchers. The faster anisotropy decay observed in the unloaded form reflects both an enhancement in the energy-transfer between the tryptophans and an acceleration of their motions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Blue copper proteins as a model for investigating electron transfer processes within polypeptide matrices.

Intramolecular long-range electron transfer (ET) processes have been investigated in two types of blue copper proteins; the single-copper protein, azurin and the multi-copper oxidase, ascorbate oxidase. These have several advantages for investigating the parameters that control the above reactions: (1) Their sole physiological role is mediating or catalyzing ET processes via the evolutionary optimized copper sites. (2) The three-dimensional structures of a considerable number of blue single copper containing proteins, e.g. azurins, and of ascorbate oxidase, have been determined at high resolution. (3) These proteins have no other cofactors except for the copper ions, thus the role of the polypeptide matrix can be addressed in a more straightforward manner. In azurins, the ET from the cystine (3-26) radical-ion produced by pulse-radiolytic reduction of this single disulfide bridge, to the Cu(II) ion bound at a distance of approximately 2.6 nm has been studied, in naturally occurring and in single-site mutated azurins. The role of changing specific amino acid residues on the internal long-range electron transfer (LRET) process and its potential pathways has been investigated. It is noteworthy that this process is most probably not part of the physiological function of azurin, hence, there has not been any evolutionary selection of structural elements for the reaction. Therefore, this provides a system for an unbiased examination of structural and chemical requirements for control of this process. By contrast, in blue copper oxidases, the internal ET from the electron uptake site from substrate to the O2 reduction site is part of these enzymes catalytic cycle, presumably optimized by selective pressure. We are investigating this internal ET in ascorbate oxidase and try to resolve the relation between this enzyme's distinct functional states and the internal ET rates. We conclude that in both types of proteins, the investigated LRET proceed primarily along covalent pathways, thus providing suitable systems where the parameters controlling the efficiency of these processes can be pursued.

Ascorbate Oxidase↗
PubMed1994

Antibodies specific to membrane components of rat mast cells are cross-reacting with human basophils.

Murine monoclonal antibodies (mAbs) have previously been raised by us with specificity to the following plasma membrane components of rat mucosal mast cells (RBL-2H3 subline): (1) the alpha-subunit of the type I Fc epsilon receptor, (Fc epsilon RI); (2) a newly described membrane glycoprotein, distinct from any of the Fc epsilon RI subunits, yet affecting the cell's secretory response to Fc epsilon RI clustering and hence named mast cell functional antigen (MAFA), and (3) a glycolipid, GD1b present in the RBL-2H3 cell's plasma membrane. The cross-reactivity of these mAbs with human basophils (from both allergic and nonallergic children) was now examined by three different protocols: (1) by microscopy (light and dark field) of double stained basophils by toluidine blue and by fluorescein-labeled anti-mouse antibodies as secondary ligands binding to the mAbs; (2) by flow cytometry (using directly labeled mAbs), and (3) by monitoring the binding of the 125I-radiolabeled mAbs. In order to exclude the possibility of the (intact) mAbs binding to the Fc gamma receptors, also present on human basophils, Fab and (Fab')2 fragments derived from the above respective mAbs were employed wherever necessary. The results show that the above described murine mAbs fragments do bind specifically to basophils obtained from allergic and nonallergic children. These antibodies may thus also be employed as tools for studying the human basophils function.

Animals↗
PubMed1993

Cloning and sequence of the cDNA coding for rat type II Fc gamma receptor of mast cells.

A clone encoding the rat type II Fc gamma receptor was isolated from a cDNA library of the rat mucosal mast cell line RBL-2H3 and its sequence determined. The predicted amino acid sequence is highly homologous with the mouse type II as well as rat type III Fc gamma receptors. The site of alternative splicing which generates the mouse Fc gamma Rb2 isoform is completely conserved. Hence we consider the new sequence to encode a rat Fc gamma RII b2 isoform. In further analogy to the mouse receptor, no consensus motif known to be involved in accessory chain association was observed in the transmembranal domain. The importance of the identification of this receptor for investigation of the immunological stimulation of mast cells is discussed.

Amino Acid Sequence↗
PubMed1993

Intramolecular electron transfer in single-site-mutated azurins.

Single-site mutants of the blue, single-copper protein, azurin, from Pseudomonas aeruginosa were reduced by CO2- radicals in pulse radiolysis experiments. The single disulfide group was reduced directly by CO2- with rates similar to those of the native protein [Farver, O., & Pecht, I. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6968-6972]. The RSSR- radical produced in the above reaction was reoxidized in a slower intramolecular electron-transfer process (30-70 s-1 at 298 K) concomitant with a further reduction of the Cu(II) ion. The temperature dependence of the latter rates was determined and used to derive information on the possible effects of the mutations. The substitution of residue Phe114, situated on the opposite side of Cu relative to the disulfide, by Ala resulted in a rate increase by a factor of almost 2. By assuming that this effect is only due to an increase in driving force, lambda = 135 kJ mol-1 for the reorganization energy was derived. When Trp48, situated midway between the donor and the acceptor, was replaced by Leu or Met, only a small change in the rate of intramolecular electron transfer was observed, indicating that the aromatic residue in this position is apparently only marginally involved in electron transfer in wild-type azurin. Pathway calculations also suggest that a longer, through-backbone path is more efficient than the shorter one involving Trp48. The former pathway yields an exponential decay factor, beta, of 6.6 nm-1. Another mutation, raising the electron-transfer driving force, was produced by changing the Cu ligand Met121 to Leu, which increases the reduction potential by 100 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Azurin↗
PubMed1993

86Rb+ ion fluxes in resting and immunologically stimulated mucosal mast cells.

We studied fluxes of Rb+ ions, using its 86Rb isotope as a radioactive tracer in living rat mucosal mast cell cultures (RBL-2H3 line) grown to high density on beads. Continuously perfused samples of these cells could be immunologically stimulated by antigen clustering of IgE bound to the cells type I Fc epsilon receptors (Fc epsilon RI) and both the cellular response, as measured by the secreted mediators, as well as the uptake of 86Rb+ of the perfused sample could be monitored. The following results were obtained. (i) In resting cells, 86Rb+ influx is observed upon exposure to extracellular 86Rb+. It proceeds with a monoexponential time course (tau = 30.6 +/- 8 min) reaching a steady-state distribution of [86Rb+]int/[86Rb+]ext = 31.6 +/- 6.4 and can be inhibited by ouabain. (ii) Fc epsilon RI clustering-mediated stimulation of these cells causes an immediate and marked increase in both amplitude and rate of 86Rb+ uptake, which also fits a monoexponential function (tau = 26.8 +/- 8.6 min). (iii) This stimulated 86Rb+ uptake can also be inhibited by ouabain. It is not caused by Ca2+ influx or by the exocytotic process as evidenced by the fact that it is also observed in buffer to which no Ca2+ ions were added. Analysis of these results by a simple model taking into account unidirectional 86Rb+ influx by the Na+/K(+)-dependent ATPase and its efflux by K+ channels yields a resting cells unidirectional K+ uptake of 3.0 +/- 1.1 10(7) ions/cell/s, which is increased by ca. 10% upon clustering of the Fc epsilon RI by IgE and antigen. The stimulated influx is suggested to be due to enhanced activity of the Na+/K(+)-dependent ATPase, reflecting increased permeability for Na+ ions.

Animals↗
PubMed1993

Distribution of type I Fc epsilon-receptors on the surface of mast cells probed by fluorescence resonance energy transfer.

The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptor aggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components.

Animals↗
PubMed1992

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.

Azurin↗
PubMed1992

Potent block of Cl- channels by antiallergic drugs.

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p < 0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.

Animals↗
PubMed1992

Fc epsilon receptor mediated Ca2+ influx into mast cells is modulated by the concentration of cytosolic free Ca2+ ions.

The relationship between the Fc epsilon receptor mediated stimulation of mast cells and the Ca2+ signal it induces were studied using thapsigargin (TG), a blocker of the endoplasmic reticulum Ca2+ pump. TG induced, in mucosal mast cells (RBL-2H3 line), a dose-dependent and an InsP3-independent increase in [Ca2+]i (from resting levels of 83-150 nM to 600-680 nM), and a secretory response amounting to 30-50% of that observed upon Fc epsilon RI clustering. The TG induced rise of [Ca2+]i is most probably provided by both arrest of its uptake by the endoplasmic reticulum and influx from the medium. Thus, Ca2+ influx in mast cells may be modulated by the [Ca2+]i level.

Animals↗