Jacqueline Keyhani

Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells. Function of copper in the assembly of active cytochrome c oxidase.

Abstract 1. 1. Cytochrome c oxidase from Candida utilis yeast cells grown in copper deficient and copper sufficient media has been studied by labeling the apoprotein with radioactive leucine ( 3 3H- and 14 C-labeled leucine). The six subunits of cytochrome c oxidase were found to be present in copper deficient cells. 2. 2. The kinetics of reconstitution of cytochrome c oxidase when copper deficient cells are grown in copper supplemented medium suggest that an active process is involved in the transfer and/or integration of copper into cytochrome c oxidase. 3. 3. When copper deficient cells are grown in copper supplemented media containing cycloheximide so that no cytoplasmic protein synthesis occurs, cytochrome c oxidase reconstitution is totally inhibited. 4. 4. When copper deficient cells are grown in copper supplemented media containing either chloramphenicol which blocks mitochondrial translation, or ethidium bromide which inhibits mitochondrial transcription, cytochrome c oxidase reconstitution is 20–25% of the control value. 5. 5. The data show that: (a) the apoprotein of cytochrome c oxidase is synthesized in copper deficient cells; (b) the insertion of copper is not necessary for the integration of the apoprotein into the membrane; (c) the transfer of copper and/or its integration into cytochrome c oxidase necessitates a cytoplasmic protein which is sensitive to cycloheximide. Thus copper plays a key role in the assembly of active cytochrome c oxidase.

Epr study of the effect of formate on cytochrome c oxidase

Abstract The addition of formate to oxidized cytochrome c oxidase (ferrocytochrome c : oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome c oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome a 3. Data suggest that binding of formate to oxidized cytochrome c oxidase produces a ligand- a 3 interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome a . Thus both cytochrome a and a 3 contribute to the resonance of g = 3 signal of cytochrome c oxidase.

HETEROGENEOUS INHIBITION OF HORSERADISH PEROXIDASE ACTIVITY BY CADMIUM

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.

Antioxidant Enzymes during Hypoxia–Anoxia Signaling Events in Crocus sativus L. Corm

Abstract:  The activity of reactive oxygen species (ROS)‐scavenging enzymes, catalase, superoxide dismutase (SOD), glutathione peroxidase, o‐dianisidine and ascorbate peroxidases, was investigated in Crocus sativus L. corms cultivated in normoxic and hypoxic–anoxic conditions. The activity of the ROS‐scavenging enzymes studied varied during cultivation. However, the pattern of ROS‐scavenging enzymes production was different in corms cultivated in normoxic and hypoxic–anoxic conditions. In normoxic conditions, only the activities of peroxidases and SOD were stimulated. In dormant corms placed under hypoxia–anoxia, the activities of catalase, SOD, and glutathione peroxidase were stimulated, with the highest stimulation observed for catalase, followed by SOD, and then glutathione peroxidase. In corms that had been rooted for 3 days before being placed in hypoxia–anoxia, the activities of all ROS‐scavenging enzymes studied were stimulated with the highest stimulation still observed for catalase, followed by the peroxidases, and finally SOD. Thus catalase was the prevailing enzyme produced under hypoxia–anoxia.

DNA Strand Breaks by Metal-Induced Oxygen Radicals in Purified Salmonella typhimurium DNA

Abstract:  Purified Salmonella typhimurium DNA was incubated for 1h at 37°C with various concentrations (10–100 μM) of transition metal ions (Fe2+, Fe3+, Cu2+, Ni2+, Cd2+), with various concentrations (0.1–100 mM) of H2O2, and with various concentrations of each transition metal ion in the presence of various concentrations of H2O2. Damage to DNA was assessed by electrophoresis of the reaction mixtures in 1% agarose gel. Breakage of the DNA strands would produce a series of DNA fragments resulting in a smear in the gel, while intact DNA produced a single band. Results showed that no damage to the DNA was detectable after incubation with either H2O2 alone or either of the metal ions alone. However, all of the metal ions investigated triggered DNA breakage in the presence of H2O2. The extent of breakage depended on the metal ion and on its concentration, as well as on the H2O2 concentration. Addition of either EDTA or catalase to the reaction mixture completely inhibited the DNA degradation, confirming the involvement of both the metal ion and the H2O2 in the breakage of DNA strands. Production of the hydroxyl radical when H2O2 and a metal ion were both present in the reaction mixture was evidenced by the thiobarbituric acid method. The most extensive damage was caused by Cu2+ followed, in decreasing order, by Fe2+, Fe3+, Ni2+, and Cd2+.

Acriflavine-mediated apoptosis and necrosis in yeast Candida utilis.

Acriflavine is an antiseptic, fungicide, and effective agent against parasitic infections, inducing petite mutation in the yeast Saccharomyces cerevisiae and kinetoplast loss in Trypanosomidae. Here we showed that acriflavine caused both apoptosis and necrosis in the yeast Candida utilis. Cells were cultured in minimal medium, with 1.5% ethanol as substrate, in the presence of 30–180 μmol/L acriflavine. Fluorescence measurements showed a linear concentration–dependence flux of the drug into the cells. Acriflavine induced a decrease in cell number, an increase in trypan blue–positive cells, and a decrease in cell viability. Cells cultured in the presence of acriflavine showed an alteration in their respiratory control ratio and a decrease in their cytochrome content. Fluorescence microscopy, after acridine orange staining, revealed the presence of apoptotic cells in cultures conducted in the presence of acriflavine. Electron microscopy of cells grown in the presence of acriflavine showed apoptotic cells exhibiting chromatin condensation, cytoplasmic lysis, but reasonably well‐preserved mitochondria, whereas necrotic cells showed no distinctive intracellular organelles. Data showed that acriflavine caused both apoptosis and necrosis. Moreover, acriflavine induced oxidative phosphorylation uncoupling. Generally, apoptosis is considered to be mediated either by a change in mitochondrial permeability and cytochrome c release or by plasma membrane death receptor activation. The outer mitochondrial membrane permeability to cytochrome c, with efflux of protons to the cytosol and cytoplasmic acidification, produced a collapse in the electrochemical proton gradient, a decrease in ATP synthesis, and subsequent cytolysis leading to apoptosis and necrosis.

Structural and Functional Alterations of Catalase Induced by Acriflavine, a Compound Causing Apoptosis and Necrosis

Acriflavine is an antiseptic agent causing both apoptosis and necrosis in yeast. In this work, its effect on the structure and function of catalase, a vital enzyme actively involved in protection against oxidative stress, was investigated. In vitro kinetic studies showed that acriflavine inhibited the enzymatic activity in a competitive manner. The residual activity detectable after preincubation of catalase (1.5 nmol/L) with various concentrations of acriflavine went from 50% to 20% of the control value as the acriflavine concentration increased from 30 to 90 μmol/L. Correlatively with the decrease in activity, alterations in the enzymes conformation were observed as indicated by fluorescence spectroscopy, circular dichroism spectroscopy, and electronic absorption spectroscopy. The enzymes intrinsic fluorescence obtained upon excitation at either 297 nm (tryptophan residues) or 280 nm (tyrosine and tryptophan residues) decreased as a function of acriflavine concentration. Circular dichroism studies showed alterations of the protein structure by acriflavine with up to 13% decrease in α helix, 16% increase in β‐sheet content, 17% increase in random coil, and 4% increase in β turns. Spectrophotometric studies showed a blueshift and modifications in the chromicity of catalase at 405 nm, corresponding to an absorbance band due to the enzymes prosthetic group. Thus, acriflavine induced in vitro a profound change in the structure of catalase so that the enzyme could no longer function. Our results showed that acriflavine, a compound producing apoptosis and necrosis, can have a direct effect on vital functions in cells by disabling key enzymes.

Hypoxia/anoxia as signaling for increased alcohol dehydrogenase activity in saffron (Crocus sativus L.) corm

Abstract: Alcohol dehydrogenase, NAD‐dependent lactate dehydrogenase, and NAD‐independent lactate dehydrogenase activities were investigated in corms cultivated in normoxic and hypoxic/anoxic conditions. Depending on the developmental stage, hypoxia/anoxia was a signal for increase in either alcohol dehydrogenase or NAD‐dependent lactate dehydrogenase. NAD‐independent lactate dehydrogenase contributed to the recycling of lactate, thus preventing acidosis.

Plasma Membrane Alteration Is an Early Signaling Event in Doxorubicin-Induced Apoptosis in the Yeast Candida utilis

Abstract: Signaling pathways such as increased ceramide, mitochondrial dysfunction, and P3 and caspase activation are produced by anticancer drugs and lead to apoptosis. In this research we show that the first event after culturing the yeast Candida utilis in the presence of low doses of doxorubicin (25 μg/mL) is the morphological alteration of the plasma membrane. In the presence of higher doxorubicin doses (≥50 μg/mL), in addition to profound alterations in the plasma membrane, changes in mitochondrial shape and cristae organization were observed. Concomitantly, increases in respiration, substrate oxidation, and cytochrome biosynthesis were observed at low doxorubicin doses (up to 25 μg/mL), whereas a progressive decrease was observed at higher doses. [3H]Leu incorporation into proteins increased by 40% in the mitochondrial fraction and by 19% in the cytosol in the presence of 25 μg/mL doxorubicin; it decreased to 80% of the control in the cytosol in the presence of 1 mg/mL doxorubicin. Morphologically, doxorubicin doses of up to 200 μg/mL produced apoptosis, whereas higher doxorubicin doses produced necrosis.

DNA-mobility shift assay and the detection of anti-DNA IgG in systemic lupus erythematosus patients.

Although the presence of antibodies against double-stranded (ds) DNA is highly specific of systemic lupus erythematosus (SLE), it is not detected in all SLE patients, perhaps due to a lack of sensitivity of the tests routinely used to assay anti-(ds) DNA. Looking for an alternative assay, this study explored the applicability of a DNA-mobility shift assay for the detection of anti-(ds) DNA; furthermore, the study compared the use of Salmonella typhimurium DNA with that of calf thymus DNA in the assay. After electrophoresis, samples containing S. typhimurium DNA and IgG from SLE sera showed marked alterations in DNA electrophoretic mobility when compared to DNA alone. In our sampling, SLE patients who tested negative for anti-(ds) DNA antibodies with routinely used assays such as Crithidia luciliae immunofluorescence test, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), tested positive for anti-(ds) DNA with the DNA mobility shift assay using S. typhimurium DNA. Incubation with IgG from control sera in the same proportions as above did not affect S. typhimurium DNA electrophoretic mobility. When S. typhimurium DNA was replaced by calf thymus DNA, the effect on the DNA mobility was less pronounced and less reliable. These results indicated that a DNA-mobility shift assay would be a useful alternative for the unequivocal detection of abnormal titers of anti-(ds) DNA antibodies. Furthermore, data indicated a greater ability of the IgG from SLE patients to form complexes with S. typhimurium DNA than with calf thymus DNA, suggesting an alternative testing DNA which may lead to a more sensitive anti-(ds) DNA detection.