Uptake and metabolic effects of insulin mimetic oxovanadium compounds in human erythrocytes

The uptake of the oxidation products of two oxovanadium(IV) compounds, [N,N'-ethylenebis(pyridoxylaminato)]oxovanadium(IV), V(IV)O(Rpyr(2)en), and bis-[3-hydroxy-1,2-dimethyl-4-pyridinonato]oxovanadium(IV), V(IV)O(dmpp)(2), by human erythrocytes was studied using (51)V and (1)H NMR and EPR spectroscopy. V(IV)O(Rpyr(2)en) in aerobic aqueous solution is oxidized to its V(V) counterpart and the neutral form slowly enters the cells by passive diffusion. In aerobic conditions, V(IV)O(dmpp)(2) originates V(V) complexes of 1:1 and 1:2 stoichiometry. The neutral 1:1 species is taken up by erythrocytes through passive diffusion in a temperature-dependent process; its depletion from the extracellular medium promotes the dissociation of the negatively charged 1:2 species, and the protonation of the negatively charged 1:1 species. The identity of these complexes is not maintained inside the cells, and the intracellular EPR spectra suggest N(2)O(2) or NO(3) intracellular coordinating environments. The oxidative stress induced by the oxovanadium compounds in erythrocytes was not significant at 1mM concentration, but was increased by both vanadate and oxidized V(IV)O(dmpp)(2) at 5mM. Only 1mM oxidized V(IV)O(dmpp)(2) significantly stimulated erythrocytes glucose intake (0.75+/-0.13 against 0.37+/-0.17mM/h found for the control, p<0.05).     

EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

Superoxide (O???) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(?-) radicals in biological systems is limited due to inefficiency of O??? spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O??? in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O???, producing stable nitroxides and allowing site-specific cO??? detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O???. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O??? both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O??? stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O???. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O??? radical in extracellular or intracellular compartments, cytoplasm or mitochondria.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.     

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

Participation of oxygen in the bacterial transformation of 2,4,6-trinitrotoluene

The exposure of Bacillus cereus ZS18 cell suspensions to 2,4,6-trinitrotoluene (TNT) in the absence of other oxidizable substrates increases oxygen uptake, exceeding the basal level of respiration of the bacterium 1.5- and 2-fold with 50 and 100 mg/liter of TNT, respectively. The interaction of both living and to less extent dead bacterial cells with TNT results in the accumulation of superoxide anion (O2*-) in the extracellular medium, which was revealed by the EPR spectroscopy. The accumulation of O2*- decreased by 50-70% in the presence of Cu,Zn-superoxide dismutase of animal origin. In the presence of living bacterial cells, the level of TNT decreased progressively, yielding hydroxylaminodinitrotoluenes together with O2*-. In the presence of heat-killed cells, a moderate decrease in TNT was observed, and the appearance of O2*- was not accompanied by the production of any detectable TNT metabolites. Chelating agents inhibited the transformation of TNT and decreased the formation of O2*-. The demonstrated generation of O2*- during the interaction of TNT with K4[Fe(CN)6] together with the observed effects of chelating agents suggest the participation of iron in the one-electron reduction of TNT and the functioning of an extracellular redox cycle with the involvement of molecular oxygen.     

An evaluation of paramagnetic broadening agents for spin probe studies of intact mammalian cells.

Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.     

The photorelease of nitrogen monoxide (NO) from pentacyanonitrosyl coordination compounds of group 8 metals.

The photodetachment of NO from [M(II)(CN)5NO]2- with M = Fe, Ru, and Os, upon laser excitation at various wavelengths (355, 420, and 480 nm) was followed by various techniques. The three complexes showed a wavelength-dependent quantum yield of NO production Phi(NO), as measured with an NO-sensitive electrode, the highest values corresponding to the larger photon energies. For the same excitation wavelength the decrease of Phi(NO) at 20 degrees C in the order Fe > Ru > Os, is explained by the increasing M-N bond strength and inertness of the heavier metals. Transient absorption data at 420 nm indicate the formation of the [M(III)(CN)5H2O]2- species in less than ca. 1 micros for M = Fe and Ru. The enthalpy content of [Fe(III)(CN)5H2O]2- with respect to the parent [Fe(II)(CN)5NO]2- state is (190 +/- 20) kJ mol(-1), as measured by laser-induced optoacoustic spectroscopy (LIOAS) upon excitation at 480 nm. The production of [Fe(III)(CN)5H2O]2- is concomitant with an expansion of (8 +/- 3) ml mol(-1) consistent with an expansion of the water bound through hydrogen bonds to the CN ligands plus the difference between NO release into the bulk and water entrance into the first coordination sphere. The activated process, as indicated by the relatively strong temperature dependence of the Phi(NO) values and by the temperature dependence of the appearance of the [Fe(III)(CN)5H2O]2- species, as determined by LIOAS, is attributed to NO detachment in less than ca. 100 ns from the isonitrosyl (ON) ligand (MS1 state).     

Binding of isotopically labeled substrates, inhibitors, and cyanide by protocatechuate 3,4-dioxygenase

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chem. 259, 4487-4495). Hyperfine broadening from [13C]cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from [13C]cyanide, but not from [17O]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by substrates and for a small molecule binding site away from the iron in intradiol dioxygenases.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

Reactions of sulfur-nitrosyl iron complexes of "g=2.03" family with hemoglobin (Hb): kinetics of Hb-NO formation in aqueous solutions.

NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (VIII) have been investigated. The method for research has been based on the formation of Hb-NO adduct upon the interaction of hemoglobin with NO generated by complexes I-VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I-VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes (I-VI) with Hb is limited by the stage of NO release in the solution.     

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with inorganic redox partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+

Plastocyanin (PCu) from spinach leaves has been singly NO2-modified, purified by FPLC, and the position of modification at Tyr83 confirmed by trypsin digestion and amino-acid sequencing. Electron-transfer reactions of native and NO2-modified PCu with the inorganic redox partners [Fe(CN)6]3- and [Co(phen)3]3+, as oxidants for PCu(I), and [Fe(CN)6]4- and [Co(phen)3]2+ as reductants for PCu(II), have been studied as a function of pH. The acid dissociation constant for the phenolic group on NO2-Tyr83 PCu is 8.78 (average) for reduced, and 8.10 for oxidised protein, as compared to values greater than 10 for native protein. At I = 0.10 M (NaCl) NO2-modification brings about a 20 mV increase in reduction potential at pH less than 7 and deprotonation of the phenolic group a 20-25 mV decrease, both transmitted to and effective at the active site. Deprotonation brings about a 48% increase in rate for [Fe(CN)6]3- and a 47% decrease for [Fe(CN)6]4- in accordance with these changes. In the case of [Co(phen)3]3+, which reacts substantially at the remote site in the vicinity of Tyr83, the influence of deprotonation on the active site is supplemented by the negative charge of the phenolate, and a total increase of 131% is observed. These results can be understood on the basis of the electron-transfer theory, and add support to the belief that electron transfer kinetics of negatively and positively charged reactants are dominated by different sites on PCu for electron transfer, namely adjacent (close to His87) and remote (close to Tyr83), respectively.     

EPR and ENDOR studies of cryoreduced compounds II of peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

Intraphagosomal oxygen in stimulated macrophages

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO₂ in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d₁₇-1-oxyl iodide (d-Cat₁)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl (¹⁵N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley-Liss, Inc.     

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Beta1-integrins mediate Ca2+-signalling and T cell spreading via divergent pathways.

Interaction of Jurkat T-lymphocytes with two extracellular matrix (ECM) proteins of the basement membrane, laminin or collagen type IV, combined with poly-L-lysine resulted in a strong adhesion, a highly increased intracellular Ca2+-concentration ([Ca2]i), as compared to cells on laminin or collagen type IV alone and in spreading of the cells. The strong adhesion was independent of an increase in [Ca2+]i, was not mediated by a beta1-integrin, and was due to charge interaction between the positively charged polyaminoacid and the negatively charged cell surface. The latter was confirmed by substitution of poly-L-lysine by other positively charged polyaminoacids. In contrast, Ca+-signalling and spreading of the cells adhering to laminin or collagen type IV combined with poly-L-lysine was completely blocked by anti-beta1 mAb. However, spreading of the cells was independent of an increase in [Ca2+]i suggesting divergent signal transduction pathways leading to Ca2+-signalling and spreading of the cells. We elucidated these signal transduction pathways by inhibition of key enzymes involved. The tyrosine kinase inhibitor genistein blocked Ca2+-signalling as well as spreading, whereas inhibitors of PKC (calphostin C, GF109203x), PLCgamma (U73122) and PLA2 (bromophenacyl-bromide (BPB), 3-[4-octadecyl)benzoyl]acrylic acid (OBAA)) selectively blocked spreading of the cells.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5

Previous M?ssbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel M?ssbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.