Modification of the structural and redox properties of cytochrome c by heteropolytungstate binding

Complex formation between horse heart ferricytochrome c and large three-dimensional polyanions has been investigated, in order to study the influence of surface electrostatic interactions on the structural and redox properties of cytochrome c. Cytochrome c binds the large heteropolytungstates (NaSb9W21O86)18- and (KAs4W40O140)27- with a 1/1 polyanion/cytochrome c ratio, and the smaller ion (SiW11O39)8- with a 2/1 ratio. Upon complexation, cytochrome c undergoes structural changes that are dependent on the size and charge of the polyanion, and on the pH and ionic strength of the medium. Three different forms of complexed cytochrome c have been characterized by optical and EPR spectroscopies, in the pH range 6.5-8: an N form, close to the native structure, an A form, analogous to cytochrome c in acidic medium, and a novel B form in which the heme pocket is open but the iron remains low-spin. The redox potential of cytochrome c is lowered to 250-220 mV (vs. NHE) in the N form, and to 80 mV in the B form.     

Modification by homocysteine thiolactone affects redox status of cytochrome C

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.     

Amifostine has antiangiogenic properties in vitro by changing the redox status of human endothelial cells

Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H₂O₂). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H₂O₂ scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and α-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H₂O₂ or by modulating NO production.     

Amifostine has antiangiogenic properties in vitro by changing the redox status of human endothelial cells

Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H2O2). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H2O2 scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and alpha-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H2O2 or by modulating NO production.     

Modification of electrokinetic properties of nuclei in human buccal epithelial cells by electric fields

The influence of an alternating (50 Hz) electric field (5--110 V/cm) on the state of human buccal epithelium cells was studied by the methods of intracellular microelectrophoresis, heterochromatin staining with orcein, and indigo carmine staining for viability and membrane integrity evaluations. Electric field exposure induced an increase in electrophoretic mobility of cell nuclei, decreased numbers of heterochromatin granules near the inner membrane of cell nucleus, and induced cell membrane damage; but cell viability was conserved. Nuclear and cell membrane properties varied with electric field strength and age of the donors. The data obtained are interpreted as evidence of electric field induced activation of the functional state of nuclei.     

Modification of carboplatin by Jurkat cells

Using [(1)H,(15)N] heteronuclear single quantum coherance (HSQC) NMR and (15)N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt(2+) anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k(1)=k(c)[CO(3)(2-)]+k(m)+k(u)N, where k(c) is the rate constant for reaction of 1 with carbonate in the medium, k(m) is the rate constant for reaction of 1 with all other components of the medium, and k(u) is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells.     

Circular dichroism and redox properties of high redox potential ferredoxins

The circular dichroism (CD) spectra of 13 examples of high-potential iron-sulfur proteins (HiPIPs), a class of [4Fe-4S] ferredoxins, have been determined. In contrast to the proposal of Carter [Carter, C. W., Jr. (1977) J. Biol. Chem. 252, 7802-7811], no strict correlation between visible CD features and utilization of the [4Fe-4S]2+/[4Fe-4S]3+ oxidation levels was found. Although most HiPIPs have these features, the model requires their presence in all species. There is also no simple relationship between CD spectral features and the presence of conserved tyrosine-19. In addition, no apparent correlation between CD properties and oxidation-reduction potential could be detected. However, amino acid side chains in close contact to the iron-sulfur cluster appear to be important in modulating spectral and oxidation-reduction properties. In particular, the negative shoulder at 290 nm and negative maximum at 230 nm correlate with the presence of Trp-80 (Chromatium vinosum numbering). Two HiPIPs that do not have Trp at this position have positive bands at 290 and 230 nm. These bands in the Ectothiorhodospira halophila HiPIPs are apparently associated with Trp-49, which is located on the opposite side of the effective mirror plane of the cluster from Trp-80. The effect of pH on circular dichroism and redox potential in Thiocapsa roseopersicina HiPIP, which has a histidine at position 49, is consistent with the interaction of the side chain with the cluster. Despite specific differences in their CD spectra, the various HiPIPs studied show general similarity consistent with structural homology within this class of iron-sulfur proteins.     

Modification of carbon nanotubes with redox hydrogel: improvement of amperometric sensing sensitivity for redox enzymes

This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4'-dimethylbpy)(2)Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)(6)(3-) solution and electrochemical impedance measurements in an equimolar Fe(CN)(6)(3-/4-) solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15Omega. The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes.     

Role of iron in the interaction of red blood cells with methylglyoxal. Modification of L-arginine by methylglyoxal is catalyzed by iron redox cycling.

Diabetes mellitus is characterized by increased methylglyoxal (MG) production. The aim of the present study was to investigate the role of iron in the cellular and molecular effects of MG. A red blood cell (RBC) model and L-arginine were used to study the effects of MG in the absence and presence of iron. Intracellular free radical formation and calcium concentration were measured using dichlorofluorescein and Fura-2-AM, respectively. Effects of MG were compared to the effect of ferrous iron. Reaction of L-arginine with MG was investigated by electron spin resonance (ESR) spectroscopy and by a spectrophotometric method. MG caused an iron dependent oxidative stress in RBCs and an elevation of the intracellular calcium concentration due to formation of reactive oxygen species. Results of co-incubation of MG with ferrous iron in the RBC model suggested an interaction of MG and iron; one interaction was a reduction of ferric iron by MG. A role of iron in the MG-L-arginine reaction was also verified by ESR spectroscopy and by spectrophotometry. Ferric iron increased free radical formation as detected by ESR in the MG-L-arginine reaction; however, ferrous iron decreased it. The reaction of MG with L-arginine yielded a brown product as detected spectrophotometrically and this reaction was catalyzed at a lower rate with ferric iron but at a higher rate with ferrous iron. These data suggest that MG causes oxidative stress in cells, which is due at least in part to ferric iron reduction by MG and to the modification of amino acids e.g. L-arginine by MG, which is catalyzed by iron redox cycling.     

Control of redox properties of cytochrome c by special electrostatic interactions

An assessment is made of the proposal: electrostatic interactions between the ferric ion of oxidised cytochrome c and its haem propionate sidechains assists in determining the value of the redox potential and plays an important role in the redox state conformation change. Differences between the properties of homologous cytochromes are proposed to be due to differences associated with the charge on their haem propionates.     

The redox properties of ascorbate peroxidase

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.     

Modification by azocombination of the catalytic properties of ribonucleases

Using pancreatic RNAase and RNAase from Act. rimosus as models, the effect of modification by azocombination on the catalytic properties of enzymes were studied. It was shown that RNAases binding to soluble dextran did not cause any significant changes in their major catalytic properties, when polymeric RNA was used as a substrate. At the same time, the physico-chemical properties of the modified enzymes may result in changes in the catalytic properties in a reaction with low molecular weight substrates. Evidence for this observation can be obtained from the increase in the synthetic activity of modified pancreatic RNAase as compared to the hydrolase activity in the dinucleotide synthesis reaction.     

Modification of monoclonal antibodies by polymers possessing chelating properties

Monoclonal antibodies to heavy chains of the heart myosin were chemically modified by chelate polymers containing EDTA or DTPA residues. The modification allows binding up to 90 atoms of heavy metals (e.g. 111In, Mn2+, Cd3+) per protein globule, i.e. an increase in the specific activity of labeling 20 to 50-fold in comparison with previous methods. Specific affinity of the modified protein is preserved. Conjugates obtained may be useful for in vivo immuno-imaging.     

Up-regulation of Bcl-2 by redox signals in glomerular mesangial cells

The mediators nitric oxide (NO) and superoxide (O2-) are known to regulate cell death and survival. In mesangial cells (MC), NO induced apoptosis and in higher concentrations necrosis. Intriguingly, cogeneration of NO and O2- in a balanced ratio promoted cell protection. Under these conditions, we noticed the accumulation of the anti-apoptotic protein Bcl-2. Its up-regulation is based on an increase in mRNA and protein level. To investigate whether oxidative stress elicits Bcl-2 expression in general, we further used the chemically unrelated oxidative agents diamide and butyl hydroperoxide. Both stimulated mRNA and protein up-regulation of Bcl-2. But in contrast to diamide, butyl hydroperoxide evoked apoptosis and necrosis despite Bcl-2 accumulation. As diamide was non-toxic, we used diamide as a Bcl-2 activator to protect MC against a subsequent toxic dose of NO. We conclude that redox changes promote Bcl-2 up-regulation that may confer cellular protection towards apoptosis.     

Modification of radiosensitivity of mammalian cells by cyclic nucleotides

Some $\text{in vitro}$ and $\text{in vivo}$ studies suggest that adesosine 3′,5′-cyclic monophosphate (cyclic AMP) may be one of the important factors in determining the radiosensitivity of certain mammalian cells; however, the role of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in radiosensitivity of mammalian cells is completely unknown. Recent data also suggest that the mechanism of radiation protection afforded by moderate hypoxia and SH-containing compounds may involve an alteration in the intracellular level of cyclic AMP. At least one $\text{in vivo}$ study shows that cyclic AMP protects hair follicles and gut epithelial cells against radiation damage; however, it does not protect lymphosarcoma and breast carcinoma in mice. If a similar phenomenon is found in humans, an elevation of the intracellular level of cyclic AMP during radiation exposure may improve the effectiveness of radiation therapy in those cases where the radiation damage of normal tissue becomes the limiting factor for a continuation of the therapy program. More $\text{in vitro}$ and $\text{in vivo}$ studies on normal and cancer cells are needed to substantiate the role of cyclic nucleotides in radiosensitivity.     

Modification of membrane physical properties, biological response and insulin binding in Friend cells by low serum concentration

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.     

Modification of blood-borne arrest properties of lymphoma cells by inhibitors of protein glycosylation suggests the existence of endogenous lectins

The requirement for intact carbohydrates of glycoproteins at the cell surface was investigated after treatment of lymphoma cells with compounds which interfere at different steps in N-linked glycosylation: swainsonine and 1-deoxynojirimycin act at different levels during the processing, so that complex oligosaccharides cannot be formed; 2-deoxyglucose, beta-hydroxynorvaline, and tunicamycin completely prevent the formation of N-linked (high-mannose as well as complex) oligosaccharides. The role of sialic acid was investigated by treating the cells with neuraminidase. These treatments resulted in altered patterns of surface-labelled glycoproteins after SDS-polyacrylamide gel electrophoresis. Blood-borne arrest of lymphoma cells in the spleen was sensitive to neuraminidase and to treatments interfering with the processing of complex N-linked oligosaccharides. It is suggested that carbohydrates are signals for cellular interactions involved in the recirculation and homing behaviour of lymphoid cells and probably interact with endogenous lectins at their site of homing.