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Biomedical subjects

I Aviram

Publications and source records attributed to I Aviram.

At least 37 records · Page 2Linked to original sources
PubMed1989

Kinetics of cell-free activation of neutrophil NADPH oxidase. Effects of neomycin and guanine nucleotides.

The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.

Enzyme Activation↗
PubMed1989

Inositol lipids and phosphatidic acid inhibit cell-free activation of neutrophil NADPH oxidase.

The effect of inositol lipids on the SDS-initiated cell-free activation of NADPH oxidase in membranes of human neutrophils was investigated. In a system consisting of low density membranes, cytosol and SDS, low doses of phosphatidylinositol, phosphatidylinositol mono- and biphosphates and phosphatidic acid interfered with activation of the oxidase. The inhibition was relieved by increasing concentrations of the cytosol. Conversely, preincubation of multilamellar phosphoinositide vesicles with cytosol reduced its ability to support activation of the oxidase.

Cardiolipins↗
PubMed1989

Defective leukocyte fungicidal activity in end-organ resistance to 1,25-dihydroxyvitamin D.

Recent studies have shown 1,25(OH)2D3 receptor-mediated modulation of leukocyte proliferation, differentiation, and function. We examined the phagocytosis and killing of microorganisms by neutrophils and monocytes from five patients of three families with hereditary resistance to 1,25(OH)2D3. Phagocytosis of microorganisms by patients' neutrophils and monocytes was normal. However, defective neutrophil killing activity toward Candida albicans (30-40% of controls) was found in all patients. The killing of Staphylococcus aureus was normal. The neutrophil chemiluminescence, nitroblue tetrazolium (NBT) dye reduction, and the generation of superoxide ions and hydrogen peroxide by neutrophils and monocytes after induction by either soluble stimuli or zymozan particles, did not differ from those in controls. The neutrophil myeloperoxidase activity was also normal. Monocytes obtained from two patients of different families before long-term calcium infusion therapy and after they became normocalcemic, demonstrated a similar impaired fungicidal activity toward Saccharomyces cerevisiae, indicating that hypocalcemia itself was not the cause of the killing defect. However, the addition of the Ca+2 ionophore A23187 (1 microM) to the test medium restored the monocyte fungicidal activity to normal. As patients' neutrophil cytosolic free calcium concentration was similar to that in controls, it is suggested that 1,25-(OH)2D3 exerts its effect on leukocyte function by a putative receptor-mediated regulation of subcellular calcium localization which may be important for fungicidal activity.

Calcitriol↗
PubMed1987

Fluoride-mediated activation of guinea pig neutrophils.

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.

Animals↗
PubMed1986

Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite.

The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 X 10(6) M-1 X S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 X 10(6) M-1 X S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.

Chloromercuribenzoates↗
PubMed1986

Activation-dependent redistribution of cellular components alters susceptibility of human neutrophils to cross-linking agents.

Contrary to resting cells, neutrophils stimulated with concanavalin A resist inhibition by bifunctional N-hydroxysuccinimide esters. Con A prestimulated, cross-linker-treated cells released superoxide upon restimulation with PMA but did not respond to chemotactic peptides. Although rates of PMA-elicited NADPH oxidase activity were lowered by the treatment, the activation parameters, namely lag times of the reaction, were not altered. The protection by Con A against blockade by cross-linkers developed concomitantly to the activation of NADPH oxidase and indicated redistribution of cross-linker-susceptible cellular components responsible for activation with PMA. The identity of these components is discussed.

Concanavalin A↗
PubMed1985

Activation of guinea-pig and bovine neutrophil NADPH oxidase by N,N'-dicyclohexylcarbodiimide.

Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the composition of the medium was investigated. At 37 degrees C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0 degree C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37 degrees C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. Km values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 microM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C3-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracycline-loaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.

Animals↗
PubMed1984

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins. A study with crosslinkers.

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.

Concanavalin A↗
PubMed1984

Reversible blockade of the respiratory burst in human neutrophils by a cleavable cross-linking agent.

Human neutrophils treated with low concentrations of the homobifunctional cross-linking reagents disuccinimidyl suberate and dithiobis(succinimidylpropionate) failed to generate superoxide in response to any of several stimuli, including phorbol myristate acetate, fMet-Leu-Phe, A23187, fluoride, and opsonized zymosan. The cross-linking reagent interfered with the activation of NADPH oxidase, but not with its activity. Cells treated with succinimidyl butyrate, a monovalent analog of the cross-linkers, underwent a normal respiratory burst. Cross-linking also inhibited degranulation, phagocytosis, and fluorescence responses of potential-sensitive dyes but had little effect on lactate production, sugar transport, the binding of fMet-Leu-Phe, or the activity of various enzymes in the cross-linked neutrophils. Most of the cellular functions inhibited through the reaction of neutrophils with the cleavable cross-linker dithiobis(succinimidylpropionate) could be restored by reduction of the disulfide bonds of the cell-bound cross-linker with dithiothreitol.

Cross-Linking Reagents↗
PubMed1983

Activation of the respiratory burst of guinea pig neutrophils by dicyclohexylcarbodiimide.

DCCD activates the respiratory burst in guinea pig peritoneal neutrophils. The onset of the superoxide producing activity is preceeded by a lag, inversely proportional to the dose of the stimulant and to the temperature. Initial rates of superoxide formation exhibit different dependencies on the concentrations of DCCD and on temperature. Activation of NAD(P)H oxidase is inhibited by preincubation of neutrophils with 2-deoxyglucose and does not require the presence of extra cellular Ca2+.

Animals↗
PubMed1981

Stepwise modification of the electrostatic charge of cytochrome c. Effects on protein conformation and oxidation-reduction properties.

Horse heart cytochrome c was progressively maleylated, and fractions containing increasing numbers of modified lysines were obtained. The 695 nm band was present in derivatives containing up to 14 maleylated residues. Circular dichroic spectra showed minor changes beginning with 8 substituted lysines; in derivatives with 14 or more maleylated lysines, circular dichroism indicated total disruption of the native conformation. The ionic strength dependence of the measured oxidation reduction potentials and second order rate constants of reduction with ascorbate varied as expected from application of Debye-Huckel theory to the differently charged derivatives. The thermodynamic oxidation-reduction potentials decreased with the increase in the number of negatively charged groups, in a manner similar to that observed for simple iron complexes.

Animals↗
PubMed1980

The chemical reactivity of fully maleylated cytochrome c.

The chemical reactivity of fully maleylated horse heart cytochrome c with oxidants, reductants, and iron ligands was studied in the presence and absence of MgCl2. In the absence of salt, the partly unfolded protein reacts rapidly with cyanide in the ferric state (k = 235 M-1s-1); the reaction is exothermic (delta H = -9.4 kcal/mol) and entropically unfavored (delta S = -7.0 e.u.). In 0.01 M MgCl2, the refoleded protein recovers the behavior towards cyanide of native cytochrome c. The oxidation-reduction potential of the refolded species is 190 mV. In the course of reduction of the unfolded form with dithionite, a reduced intermediate species (t 1/2 = 5.9 s) is observed; this species binds carbon monoxide rapidly, but the ligand dissociates thereafter. This shows that full maleylation does not disrupt the conformational stability of reduced cytochrome c.

Animals↗
PubMed1979

The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.

Animals↗