Irit Aviram

Structural studies of modified cytochromes c by nuclear magnetic resonance spectroscopy

Abstract The binding of methionine to one of the axial positions of the heme iron in various modified mammalian-type cytochromes c has been studied by nuclear magnetic resonance spectroscopy. The modifications of the protein included formylation of tryptophan, alkylation of methionine, and polymerization by treatment with ethanol. Comparison of the data on methionine coordination with data on the ability of the modified cytochromes c to restore the respiration of cytochrome c -depleted rat liver mitochondria, indicates that the redox carrier properties of cytochrome c are maintained only if methionine is bound to the heme iron in the oxidized and the reduced form.

The role of the lysines in the alkaline heme-linked ionization of ferric cytochrome c.

Abstract Fully amidinated cytochrome c has a 695 nm band which is lost with a pK of 9.2 in a transition to a low-spin state. Fully maleylated cytochrome c can also exist in a low-spin state without the 695 nm band although the pH of the transition is very dependent on the salt concentration. Neither of these derivatives have free lysine groups. It is concluded that the hemelinked alkaline ionization of native cytochrome c is not triggered by deprotonation of a lysine residue, and that lysine does not necessarily coordinate the iron in the low-spin alkaline species.

Modification of the tryptophanyl residue of horse heart cytochrome c.

Abstract Incubation of cytochrome c with formic acid-HCl results in modification of the single tryptophanyl residue. The ultraviolet spectrum changes, the Soret band shifts to 406 nm, and the 695 nm absorption band disappears. Concomitantly the product looses its biological activity, and characteristic hemochrome properties. Alkali treatment causes an almost complete reversion to the native molecule. In view of the marked effect caused by the modification of the tryptophanyl residue, its role in native cytochrome c is discussed.

Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes

Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X‐100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)‐anchored neutrophil protein, possibly FcγRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcγRIIIb, the β2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol‐specific phospholipase C (PI‐PLC) reduced amounts of PR3 and FcγRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcγRIIIb acts as a membrane adaptor for PR3. FcγRIIIb of HMW fractions co‐immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo‐NHS‐biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58‐lysed neutrophils were similar in their protein composition to the HMW complexes but not identical. J. Cell. Biochem.

The interaction of benzhydroxamic acid with horseradish peroxidase and its fluorescent analogs

Abstract Horseradish peroxidase (HRP) reconstituted with protoporphyrin IX or zinc protoporphyrin IX binds benzhydroxamic acid with affinities of 1.54 × 10 4 and 8 × 10 2 m −1 , respectively. This interaction is competitive with respect to hydrogen donor substrates of peroxidase. The steady-state oxidation of benzhydroxamic acid by HRP was studied by monitoring the disappearance of the hydroxamate function and the formation of nitrite. The inhibition by benzhydroxamic acid of oxidation of HRP substrates may be classified as an inhibition by a competing substrate. In the case of HRP-catalyzed oxidation of ferrocyanide a marked activating effect of benzhydroxamic acid was observed. Mechanisms responsible for this effect are discussed.

Distinction between oxidizing and reducing sites of cytochrome C by chemical modification with pyridoxal phosphate

It was recently reported that antibodies to cytochrome c inhibit selectively the oxidation of the enzyme by cytochrome oxidase and its reduction by succinic dehydrogenase [ I] . However, attempts to differentiate between oxidizing and reducing sites of cytochrome c through chemical modification have been hitherto inconclusive. In this communication, we describe the preparation of cytochrome c derivatives obtained by covalent binding of pyridoxal phosphate, with normal physicochemical properties and reactivity with cytochrome oxidase, but with impaired enzymic reducibility.

Proton magnetic relaxation and anion effect in solutions of acid ferricytochrome c.

Abstract Measurements of the longitudinal relaxation rates of water protons in aqueous solutions of ferricytochrome c and their temperature dependence, were used for the elucidation of the heme iron ligands at acid pH. The relaxation rates increased with a decrease in pH and pK values of 2.5 and 4.48 were evaluated for the aqueous and 6 m urea solutions, respectively. The results at acid pH are compatible with a structure in which two water molecules exchange rapidly between the coordination sphere of high spin heme iron and the bulk. They suggest that concomitantly with the low-high spin transition the histidine-18 and methionine-80 iron bonds break simultaneously. Addition of various anions, including methanesulfonate at pH 1.95 caused a 85% decrease in the net longitudinal relaxation rate. However, neither the chemical shift nor the width of the methyl proton nmr line of methanesulfonate in solution of acid ferricytochrome c were affected indicating that the effect of anions is not due to a direct binding to the heme iron. The relaxation mechanism of the water molecules in the first coordination sphere of the ferric ion in acid cytochrome c is discussed. It appears that the longitudial relaxation rate is modulated by the electronic correlation time of the ferric ion which was calculated to be τ s = 6 × 10 −11 sec at 60 MHz.

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.

Structure-function relationship in the interaction of mastoparan analogs with neutrophil NADPH oxidase.

Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown to block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxidase activation was now demonstrated in the semirecombinant NADPH oxidase system. The structural basis of the inhibitory effect of MP on oxidase assembly was explored employing a variety of truncated and specifically substituted synthetic peptide analogs. The data indicated that an alpha helical fold, positive net charge, hydrophobicity and amphiphilicity were essential for the inhibitory potency and that peptide analogs below eleven residues were inactive. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rac1 in conjunction with immobilized MP or soluble (125)I-tyr-MP, respectively. The data implicated p67-phox as the main MP-binding component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-phox and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a low affinity towards MP.

Stimulation of neutrophils by prenylcysteine analogs: Ca2+ release and influx

Farnesylthiosalicylic acid (FTS), a synthetic analog of the terminal prenylcysteine present in signaling proteins induces generation of superoxide ions, phospholipase C-driven hydrolysis of inositol lipids and calcium elevation in human neutrophils and DMSO-differentiated HL60 cells. These effects were ascribed to an interaction of the analog with elements responsible for recognition of specific prenylated proteins. The present study demonstrated that in addition to the release of intracellular calcium stores, FTS enhanced entry of Ca(2+) and Mn(2+) from the medium. The biphasic dependence of the influx on the concentration of FTS, as well as its insensitivity to inhibition by PMA and La(3+) suggest that the influx pathway activated by FTS is distinct from the previously described store-operated calcium channels of neutrophils. Consistent with the participation of a cellular membrane component in the interaction, FTS enhanced (45)Ca uptake in neutrophils and neutrophil cell membranes, but not in multilamellar vesicles. To establish specificity of the farnesyl moiety of FTS (C(15)), effects of three other analogs, geranylthiosalicylate, GTS (C(10)), geranylgeranylthiosalicylate, GGTS (C(20)), as well as the carboxymethyl ester FTS-Me on calcium homeostasis and superoxide production were investigated. GGTS dose-dependently elevated [Ca(2+)](i), induced quenching of the 360 nm Fura-2-calcium fluorescence by Mn(2+) and stimulated superoxide release, while GTS and FTS-Me were inactive. These results defined specific structural requirements for the functional interaction of prenylcysteine analogs with myeloid cells.