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Biomedical subjects

I Pecht

Publications and source records attributed to I Pecht.

At least 145 records · Page 8Linked to original sources
PubMed1983

Pulse radiolysis kinetics of the reaction of hydrated electrons with ferric-, ferrous-, protoporphyrin IX- and apo-myoglobin.

The kinetics of the reaction of hydrated electron (e-aq) with ferric-, ferrous-, metal-free protoporphyrin IX-and apo-myoglobin have been studied by pulse radiolysis so that a direct kinetic measure of the relative reactivities of the heme and the protein part of myoglobin can be made. The second-order association rate constant with ferric Mb is about 3-times that for ApoMb, while ferric Mb, ferrous Mb and protoporphyrin IX-Mb all react at about the same rate, indicating that it is mainly the porphyrin that is the electron-attracting site. The magnitude of the rate constants (8-25 nM-1 X S-1) indicates that the encounter of e-aq with the protein is almost certainly diffusion-controlled. The initial encounter is probably followed by electron migration along parallel paths to the heme and most likely several of the 12 histidine residues. The heme competes very effectively (approx. 70%) with these other sites. The kinetically measured reduction yield of heme is consistent with that found spectrally, indicating that a histidine radical on the protein does not effectively transfer an electron intramolecularly to the heme. The spectral changes found upon the completion of the fast reaction (approx. 40 microseconds) for protoporphyrin IX-Mb and ferrous Mb are consistent with the formation of a porphyrin anion radical. For ApoMb the spectral changes are consistent with the formation of a histidine free radical.

Animals↗
PubMed1983

Membrane potential changes during IgE-mediated histamine release from rat basophilic leukemia cells.

The membrane potential of rat basophilic leukemia cells (RBL-2H3 cell line) has been determined by monitoring the distribution of the lipophilic [3H] tetraphenylphosphonium cation between the cells and the extracellular medium. By this method, the determined potential of these cells, passively sensitized with IgE, is -93 +/- 5 mV (mean +/- SEM, interior negative). Almost 40% of this membrane potential is rapidly collapsed upon the addition of the proton carrier, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). It is suggested that the FCCP-sensitive fraction of the total membrane potential results from the accumulation of this cation by the mitochondria, which maintains a negative membrane potential. Thus, the resting plasma membrane potential of these cells equals -55 +/- 6 mV. During the process of immunological stimulation by antibodies directed against cell membrane bound IgE, the membrane potential decreases. Moreover, there is a correlation between the extent of degranulation of the cells and the depolarization. It is concluded that in common with other secretory systems, depolarization of the plasma membrane is involved in the stimulus-secretion coupling of the histamine secreting RBL cells.

Animals↗
PubMed1983

A microfluorometric assay for cholinesterases, suitable for multiple kinetic determinations of picomoles of released thiocholine.

A highly sensitive microfluorometric assay for cholinesterases has been developed. Enzymatic activity is measured by monitoring the thiocholine produced by specific hydrolysis of acetylthiocholine. This is carried out by reacting the thiocholine formed with the fluorogenic compound N-(4(7 diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide to yield an intensely fluorescent product. The assay is linear over a range extending from a few picomoles to nanomoles of thiocholine. The specificity and accuracy of this microfluorometric assay were examined using microgram quantities of rat brain tissue as a source for cholinesterases. The specific activities and the Km values determined by this new method for both cholinesterase activities present in the brain (acetylcholine hydrolase, EC 3.1.1.7, and "nonspecific" cholinesterase-acylcholine acylhydrolase, EC 3.1.1.8) were identical to those reported earlier using the less sensitive spectrophotometric and radiometric methods. The background emission caused by nonenzymatic hydrolysis of the substrate is relatively low, and does not exceed background values encountered in other methods. The assay may be used for monitoring the kinetics of enzymatic activities in microscale reaction mixtures, providing a linear determination of the thiocholine produced over a period of at least 30 h at room temperature. The method can also be adapted for use in other enzymatic assays where reagents containing thiol groups can be produced or consumed.

Acetylthiocholine↗
PubMed1983

Structure-activity relationship in the mast cell degranulating capacity of neurotensin fragments.

The mast cell degranulating capacity of neurotensin and three of its fragments was examined. In Tyrode solution (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.4 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 5.6 mM glucose, pH 7.4), neither intact neurotensin nor its C-terminal tripeptide (Tyr-Ile-Leu) caused any release of histamine. Concentrations of neurotensin exceeding 10(-4)M did cause histamine release but through lysis of the cells. The C-terminal hexa- and octapeptides of neurotensin (Arg-Arg-Pro-Tyr-Ile-Leu and Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu, respectively) induced a non-cytolytic release of histamine with the latter peptide being more active (ED50 = 90 microM for the hexapeptide and 13 microM for the octapeptide). This release was not affected by the C-terminal tripeptide. It was found to be calcium-dependent and was inhibited by the anti-allergic drug, disodium cromoglycate. Phosphatidylserine did not enhance release of histamine and saturation of the immunoglobulin E (IgE) receptors did not inhibit it.

Animals↗
PubMed1983

Restoration of Ca2+ influx and degranulation capacity of variant RBL-2H3 cells upon implantation of isolated cromolyn binding protein.

Recently, variants of the rat basophilic leukemia cells (RBL-2H3), deficient in their binding capacity for the antiallergic drug cromolyn but displaying unimpaired ability to bind IgE, were selected and cloned [Mazurek, N., Bashkin, P., Petrank, A. & Pecht, I. (1983) Nature (London) 303, 528-530]. Although the histamine content and the number of IgE receptors in these variants are similar to those of the parental cells, they cannot be stimulated immunologically to allow Ca(2+) influx and to degranulate. In contrast, the Ca(2+) ionophore A23187 causes these variants to degranulate, indicating that the mechanism distal to the Ca(2+) gating is intact in the variants. The cromolyn binding protein (CBP), present in the membranes of RBL-2H3 cells, has recently been isolated by affinity chromatography under nondenaturing conditions. In the current study we have used Sendaivirus envelopes as fusogenic carriers to implant the purified CBP into the membrane of variant basophils that were defective in it. This fusion leads to the restoration of Ca(2+) uptake and degranulation capacity of the variants after IgE-mediated stimulation. These restored activities seem to show a sigmoidal dependence on the amount of incorporated CBP. Saturation values comparable to those of the parental line are reached when the level of implanted CBP approaches its density on the latter line. The restored capacity is due to the implanted CBP, because the reinstated immunological response can be blocked by the inhibitory drug cromolyn and by monoclonal antibodies specific to CBP, both shown to prevent Ca(2+) uptake and degranulation in mast cells and parental RBL-2H3 cells. These results point out that CBP plays an important role in the Ca(2+) gating process resulting in degranulation.

Animals↗
PubMed1982

Resolution of two distinct electron transfer sites on azurin.

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.

Azurin↗
PubMed1982

Electron uptake and delivery sites on plastocyanin in its reactions with the photosynthetic electron transport system.

French bean plastocyanin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert (Cr(III) adduct at the protein surface. The effect of the modification on the activity of plastocyanin in electron transfer between photosystems II and I has been investigated. The photoreduction and photooxidation by chloroplasts or by photosystem I reaction centers, respectively, chloroplasts or by photosystem I reaction centers, respectively, of native and Cr(III)-labeled plastocyanin have been compared. It was found that whereas the photoreduction rates of native and Cr-labeled plastocyanin were indistinguishable, the rates of photooxidation of the modified protein were markedly attenuated relative to those of the native one. This difference in reactivity clearly reflects the perturbation of the electron transfer pathway to P700. These findings, in conjunction with the structure of plastocyanin and the locus of CR(III) binding on its surface, lead to the following interpretation: (a) There are most probably two physiologically significant, electron transfer sites on plastocyanin. (b) The site involved in the electron transfer to P700 is most likely in the region of tyrosine-83 and the negatively charged patch proximal to it. By elimination we assume that the second site is centered at the hydrophobic region of histidine-87.

Chloroplasts↗
PubMed1982

Isolation of a basophilic membrane protein binding the anti-allergic drug cromolyn.

The membrane protein component in basophils, responsible for the specific, Ca2+-dependent, binding of the anti-allergic drug cromolyn [disodium cromoglycate, DSCG; the disodium salt of 1,2 bis(2- carboxychromon -5- yloxy )-2-hydroxy propane] was isolated by two procedures based on affinity for the drug. In the first procedure, involving immunoprecipitation, rat basophilic leukemia cells (RBL-2H3), surface labeled by 125I were reacted with a polyvalent conjugate of DSCG and bovine serum albumin and then subjected to solubilization by the non-ionic detergent Nonidet P-40 (NP-40). From these lysates, precipitation was specifically attained by subsequent addition of rabbit anti-DSCG antibodies. In an SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single radioactive band was observed, having an apparent mol. wt. of 60 000 daltons. Competitive inhibition of the immunoprecipitation in the presence of free drug or excess of EDTA demonstrated the specificity of the isolation. Furthermore, this particular membrane component could not be isolated from several other cell types examined. The second isolation from several other cell types examined. The second isolation procedure employed affinity chromatography on DSCG immobilized on polyacryl- hydrazido agarose beads. The DSCG-binding protein was eluted from the affinity column with either DSCG or with EDTA and also migrated on SDS-PAGE as a single band of 60 000 mol. wt., similar to that obtained by the immunoprecipitation procedure. These and other results suggest that this newly isolated protein is the one responsible for the Ca2+-dependent binding of the drug to the basophil membrane.

Animals↗
PubMed1981

A heterologous immunoglobulin chain recombinant carries a distinct site for dinitrophenyl and obeys the common hapten binding mechanism.

A heterologous recombinant of the immunoglobulin alpha heavy chain derived from MOPC-460 and the lambda light chain from MOPC-315 was prepared. This H460L315 hybrid binds N epsilon-(2,4-dinitrophenyl)-L-lysine (DNPL) with an affinity of 1.6 X 10(4) M-1 (7 degrees C). This Ig-hapten complex exhibits an absorption spectrum which is different from those observed for each of its parent-DNPL complexes. Very small quenching is caused in the intrinsic fluorescence of the hybrid upon hapten binding, as contrasted by the large quenching of the parent molecules. Chemical relaxation kinetic measurements show that H460L315 exists in solution in two conformations which exchange with a relaxation time of 20 ms (7 degrees C). This transition is accompanied by a change in the fluorescence quantum yield of the protein. DNPL binds to both conformers at comparable fast, though not diffusion-controlled, rates. The equilibrium between the two conformers is shifted upon hapten binding, and the two complexes exchange at a faster rate than the free protein conformers. Thus H460L315 carries a new binding site for DNPL but follows the common mechanism of hapten binding as that observed for other immunoglobulins. These properties of the hybrid should be closely related to the interactions between its constituting domains.

Animals↗
PubMed1981

Structure and properties of 5-deazaflavin radicals as compared to natural flavosemiquinones.

In order to gain more insight into flavin radicals, on which the selection of 1e-, and 2e- -oxireduction modes in flavoproteins depends, we have investigated structure, spectral properties and decay mode of molecular species occurring in the half-reduced 5-deazaflavin "model" system by flash photolysis and pulse radiolysis. (1) Enforced 1e- -reduction of 5-deazaflavin yields the short-lived red-colored 1-HdFl, which is a strong reductant. In the absence of any electron acceptor, this radical decays by 1,5-prototropy (see below) and dismutation, which is rapidly reversed upon illumination. Competing with this photo-comproportionation, irreversible formation of the photo-stable sigma-dimmer (HdFl)2, covalently linked via C(5), is observed, which becomes prevalent under prolonged illumination. (2) Enforced 1e- -abstraction from 1,5-dihydro-5-deazaflavin yields the tautomeric 5-HdFl, which is a mild oxidant and is transparent at lambda 480 nm. Prototropy 5-HdFl in equilibrium or formed from 1-HdFl can be rate-determining in 5-deazaflavin redox reactions. Hence, the radical state in the 5-deazaflavin system does not mediate double 1e- -oxidoreduction as do natural flavosemiquinones. Instead, 5-deazaflavin flavors nucleophilic substrate addition (carbanion transfer) and formation of intermediate sigma-adducts in (photo)reductions even over the extent observed with natural flavin. This confirms the description of 5-deazaflavin as a "flavin-shaped nicotinamide derivative". It explains at the same time the mechanism of 5-deazaflavin acting as a mild and yet potent photosensitizer in 1e- -reductions of biological redox systems. (3) It is shown that replacement of N(5) by CH in the flavin nucleus also leads to the disappearance of the known action-pK in the photoreduction, which confirms the assignment of the latter pK in the natural flavin system to 5-protonation of the excited flavin triplet. From these model studies the following biological conclusions can be confirmed: The tautomer equilibrium of natural flavin semiquinones is diffusion-controlled and regulated thermodynamically: 5-HFl in equilibrium or formed from 1-HFl, while in flavo-proteins the same equilibrium is regulated by regiospecific H-bridges from the apoprotein, which thus decides between 1e- - (stable 5-HFl) and 2e- -reaction (unstable 1-HFl) modes.

Electron Transport↗
PubMed1981

Proton magnetic resonance relaxation in Pseudomonas aeruginosa cytochrome oxidase solutions.

We have measured the temperature and frequency dependence of solvent proton magnetic relaxation rates in solutions of Pseudomonas aeruginosa cytochrome oxidase (EC 1.9.3.2) in its native low spin oxidized, its reduced, and its carbonyl reduced derivative. In solutions of the native oxidized enzyme, a large paramagnetic enhancement of the proton NMR relaxation rates, propagated to the solvent by the fast exchange mechanism, is observed. The ratio (T1/T2)pmg = 1.25 +/- 0.10 at 24 MHz demonstrates that dipole-dipole interaction of the neighboring paramagnets is the dominant relaxation mechanism. Measurements of proton relaxation in solutions of cytochrome oxidase from which the hemes D have been extracted demonstrates that hemes C do not contribute to the observed paramagnetic effects. The electron spin relaxation time of the ferric hemes D of 3.2 +/- 0.4 ns is calculated from the frequency dispersion data. This is the longest value reported for hemoprotein solutions so far. These features of a low spin ferric hemoprotein are similar to those found recently both for the microbial and for the microsomal cytochrome P-450. The calculated distances between the exchanging proton(s) and heme D iron ions demonstrate the high accessibility of the environment of heme D from the solvent side, also for molecules not penetrating the inner coordination sphere.

Electron Transport Complex IV↗
PubMed1981

Identification of an electron transfer locus in plastocyanin by chromium(II) affinity labeling.

Cu(II)--plastocyanin from French beans (Phaseolus vulgaris) is reduced quantitatively by Cr(II)aq ions to give a substitution-inert Cr(III) adduct of Cu(I)--plastocyanin. Enzymatic proteolysis of this derivative by thermolysin led to the identification of the Cr(III) binding peptide. This contains four potential ligands for the metal ion: aspartate-42 and -44 and glutamate-43 and -45. In the three-dimensional fold of plastocyanin, this stretch is very close to tyrosine-83. The emission intensity and its pH dependence observed for the tyrosines in this tryptophan-devoid protein differ markedly in the Cr(III) adduct. That difference is interpreted as reflecting proximity and interaction between the latter metal ion and tyrosine-83. The distance between the copper center and the suggested Cr(III) binding site is approximately 12 A. The intervening region contains an array of highly invariant aromatic residues. These are proposed to be involved in the electron transfer process. A mechanism for that process is presented that involves interaction between the d electrons of the metal ions with d pi-pi* delocalization through a weakly coupled pi* system. The rationale of this electron transfer pathway for the reactivity of plastocyanin with inorganic redox agents is discussed.

Affinity Labels↗
PubMed1981

Electron transfer between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa.

The electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa have been studied. The equilibrium constant K = ([Cyt(III)] . [Az(I)])/([Cyt(II)] . [Az(II))]) = 0.5 at 25 degrees C is about seven times smaller than that observed between the cytochrome c551 and the titrations confirmed a 43-mV difference between the mid-point potentials of +266 mV and +309 mV for the Alcaligenes and Pseudomonas azurins respectively. The kinetics of the reaction between Alcaligenes azurin and Pseudomonas cytochrome c551 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction between P. aeruginosa azurin and cytochrome c551 is not observed with the Alcaligenes azurin. The simplest mechanism that can therefore be ascribed to the investigated system is: [formula: see text]. This scheme is similar to that proposed earlier for the reaction between P. aeruginosa azurin and cytochrome c551 but does not involve the conformational transition proposed for azurin. The specific rates for the electron transfer are still fast: 1.8 x 10(6) M-1 . s-1 and 3.0 x 10(6) M-1 . s-1 respectively at 25 degrees C.

Alcaligenes↗