Influence of pH on sulfhydryl groups and fluidity of the mitochondrial membrane

Fluidity of the red blood cell membrane decreases as pH changes from 8 to 7.5. In rat liver mitochondrial (RLM) membrane fluidity precipitously declines as pH drops from 7.35 toward 7.0. With dithionitrobenzoate (Nbs2), reaction rates of mitochondrial -SH groups from rat liver and heart (RHM) and in beef heart submitochondrial particles are reduced at pH 7.0 as compared to 7.35. Similar results are obtained with the lipophilic fluorescence dye monobromobimane (MB). Bromobimane Q (MQ), which predominantly labels superficially located -SH groups, does not detect differences in -SH reaction rate between pH 7.35 and 7.0. Oligomycin diminishes the amount of reactive -SH groups in RLM titrated with Nbs2 only at pH 7.35, whereas with MB a decrease caused by oligomycin is found at pH 7.35 and pH 7.0. With MQ, an increase in reaction rate is observed for both pH values after pretreatment with oligomycin. Using 4-maleimido-TEMPO mobilization of -SH groups is found with oligomycin at pH 7.0, whereas at pH 7.35 they are immobilized. Phosphate significantly stimulates reaction rates of -SH groups at pH 7.0 in RHM and RLM. In RHM inhibition of succinate oxidation by oxaloacetate as well as the efflux of NAD(P)H is enhanced at pH 7.0, indicating increased permeability in both directions. Decreases in pH, fluidity, and thiol reactivity are important factors in hypoxic/ischemic membrane damage.     

Effect of mercury ions on cysteine metabolism in Xenopus laevis tissues

The effect of mercury ions on the level of cysteine, glutathione, sulfane sulfur, and on the activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase in brain, heart muscle, liver, kidneys, testes and skeletal muscle of adult Xenopus laevis was investigated. Frogs of both sexes were exposed for 7 or 14 days to 1.353mgL(-1) (ppm) of mercury chloride (HgCl(2)) dissolved in water. The activity of the investigated enzymes participating in cysteine metabolism depends on cysteine in their active sites. Mercury ions can bind to -SH groups and, therefore, lower the activity of enzymes and change the level of sulfane sulfur, a product of l-cysteine desulfuration. The effect of mercury was found to depend on the time of exposure and the kind of tissue. In the liver, the main site of glutathione biosynthesis, the ratio of GSH to GSSG was essentially unchanged. The total glutathione level was decreased after 7 days of exposure to mercury, similarly as the activity of rhodanese. Sulfane sulfur levels were significantly increased after a shorter duration, while they decreased after a longer time of exposure. The kidney, brain and testes were able to enhance the level of GSH, probably thanks to high γ-glutamyltranspeptidase activity. These tissues showed an increased value of GSH/GSSG ratio during the shorter exposure to mercury. The activity of sulfurtransferases was decreased, especially after the longer exposure to mercury. In the heart and skeletal muscle, the level of GSH, sulfane sulfur, and the activity of the investigated sulfurtransferases was diminished after 14 days of exposure to Hg. It can be concluded that the main mechanism of toxic Hg activity is generation of reactive oxygen species in cells due to depleted GSH level, and a decreased sulfurtransferases activity either by blocking or oxidation of their -SH groups, what in consequence results in a diminished sulfane sulfur levels in tissues, especially the heart and testes.     

The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.     

A flow cytometric assay for intracellular nonprotein thiols using mercury orange

The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcomings of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells.     

Absorption and Fluorescence Spectra of Euchrysine Ggnx and Acridine Orange; Effects of Heparin, NaCl and a Cation Exchange Resin

The absorption and fluorescence spectra of two samples of dye labeled euchrysine were found to differ. One sample, labeled GGNX, had absorption and fluorescence maxima of 435 and 515 nanometers (nm) respectively. The other sample was not further labeled, but had absorption and fluorescence maxima of 492 and 535 nm. The latter values, as well as the shape of both the fluorescence and absorption curves of the second sample were superimposable on a recrystallized sample of acridine orange labeled correctly C. I. 46905. Euchrysine has two free amino groups which are fully methylated in acridine orange, therefore a nitrous acid test can differentiate the two dyes. The sample of euchrysine labeled GGNX gave a reaction, as did acridine yellow, C. I. 46025, but acridine orange, C. I. 46005, did not. Fluorescence metachromasy of euchrysine is less efficient than that of acridine orange in two ways: the shift in the spectrum is smaller by about 40 nm, making the separation of the colors more difficult both visually and by instruments and the metachromatic fluorescence has less than half of the intensity of acridine orange as measured at the peak for each dye. Confusion between these two dyes has occurred because suppliers have used the names interchangeably. For critical studies, the dye used should be identified by its Colour Index number.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Application of mercury cold vapor atomic fluorescence spectrometry to the characterization of mercury-accessible -SH groups in native proteins.

A new analytical approach has been applied to the determination and characterization of mercury-accessible -SH groups in pure native protein samples (ovalbumin, hemoglobin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, hexokinase, lactate dehydrogenase, alcohol dehydrogenase, creatine phosphokinase, lysozyme, and cytochrome c). The method is based on the selective reduction of Hg(II) in the presence of Hg(II)-thiol complexes with alkaline sodium tetrahydroborate, to give Hg(0) in a continuous flow reaction system coupled with atomic fluorescence spectrometric (AFS) detection. The method is fast and specific and allows one to work with nanomole amounts of a single protein without any preliminary incubation and without any separation of Hg(II) from thiol-complexed mercury. The meaning of the results obtained in the determination of the accessible -SH groups in native proteins by using chemical probes is discussed.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

Histochemical localization of glutathione in tissues.

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.     

Radio- and thermosensitivity of E. coli K1060 after thiol depletion by diethylmaleate

Summary The Escherichia coli auxotroph K1060 has been grown in a medium supplemented with either oleic acid (18 : 1) or linolenic acid (18 : 3) and its radiosensitivity and thermosensitivity established using bacterial cell survival as the assay system. No difference in radiosensitivity was observed when oleic and linolenic grown cells were exposed to-radiation at room temperature. When heated at 49° C linolenic grown cells were more sensitive than oleic grown cells.To investigate whether soluble -SH compounds, e.g., glutathione (GSH), were critical in protecting cells against radiation or heat, studies were performed using cells depleted of -SH by incubation with diethylmaleate (DEM). After reduction of water-soluble non-protein thiol compounds to 25% (10 mM DEM treatment) of control value, no major changes in radiosensitivity under oxic conditions were found. Radioresistance increased slightly when irradiation was performed under hypoxic conditions. Thermoresistance was clearly stimulated after DEM treatments between 1 and 10 mM DEM.The main conclusion of these experiments is that lowering the cellular level of reduced glutathione may not generally be correlated with a higher radio- and thermosensitivity.     

Optimization of flow-cytometric discrimination between reticulocytes and erythrocytes

An automatized technique to count reticulocytes by means of flow cytometry is described. Blood samples were stained by the fluorescent dye acridine orange without the use of fixative. Scatter and red fluorescence of the blood cells were measured in a flow cytometer. A discrimination between reticulocytes and erythrocytes was only achieved by using logarithmic amplification. The discrimination was better in peak mode than in area mode. The optimum dye concentration was 0.5 mg/liter acridine orange. At lower dye concentrations, not all reticulocytes were measured, whereas at higher dye concentrations the degree of discrimination between reticulocytes and erythrocytes decreased. There was a suitable discrimination between reticulocytes and erythrocytes. The reticulocyte numbers were scored by flow cytometry as well as by microscope for blood samples with 0.1-14% reticulocytes. The correlation between both methods was close.     

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 chinese hamster cells after treatment with colcemid,diamide, carbaryl and methyl mercury

Asynchronously growing V79 Chinese hamster cells were treated with colcemid, diamide, carbaryl and methyl mercury, which are all known to be spindle disturbing agents. For each compound the dose response for c-mitosis, survival and level of free sulfhydryl groups was investigated under comparable conditions. Diamide, carbaryl and methyl were all found to give a significant increase of c-mitosis at a dose giving a decrease of non-protein sulfhydryl groups (NPSH, mainly glutathione) of 30–40% suggesting that a decrease of this magnitude may have a predictive value for spindle disturbances. Despite this similarity at concentrations close to the respective thresholds it was found that the c-mitotic activity at higher concentrations was not a simple function of average NPSH decrease. Diamide, which rapidly oxidizes glutathione to glutathione disulfide, was a less efficient c-mitotic agent than carbaryl and methyl mercury in relation to average NPSH decrease at higher concentrations. Protein bound sulfhydryl groups (PSH) were not significantly affected with diamide and carbaryl at their lowest c-mitotic concentrations while methyl mercury caused a significant decrease already at concentrations below the lowest c-mitotic concentration. With colcemid a significant decrease of average NPSH (14%) and PSH (12%) was observed only with concentrations giving close to 100% c-mitotic cells. Concentrations giving more than 20% c-mitosis gave a pronounced decrease of survival with carbaryl, diamide and methyl mercury while no toxic effects were obtained with colcemid, not even with concentrations giving close to 100% c-mitosis. Carbaryl, diamide and methyl mercury caused increased glutathione peroxidase activity indicating that these compounds cause increased lipid peroxidation. The possible connection between peroxidative damage of membranes and c-mitosis is discussed.     

Phenylarsine oxide and the mechanism of insulin-stimulated sugar transport

The actions of phenylarsine oxide (PAO) on hormone receptors and transport processes are reviewed with particular reference to the mechanism of insulin-stimulated sugar transport. It is suggested that as well as reaction with vicinal -SH groups, vicinal -SH/-OH and -SH/-CO2H groups should also be considered as potential reaction sites for PAO. The relatively high levels of these vicinal combinations of groups in many hormone receptors makes them particularly susceptible to reaction with PAO. In the case of insulin-stimulated sugar transport PAO does not inhibit insulin binding to its receptor at low concentrations but may react directly with the glucose transporters in some cells. A hypothesis is proposed suggesting that PAO may react specifically with one transporter isoform (GLUT-4) which is found almost exclusively in rat adipocytes, skeletal muscle and heart tissue (i.e. insulin responsive tissue) whereas in insulin unresponsive cells such as erythrocytes the GLUT-1 isoform is the predominant transporter which is not inhibited by PAO.     

Y-27632 induces calcium-independent glutamate release in rat brain synaptosomes by the mechanism which is distinct from exocytosis

The inhibitor of Rho-kinase Y-27632 induces non-secretory exocytosis in PC12 cells. The influence of this compound on central synapses remains uninvestigated. We showed that Y-27632 at the concentration 100 jtM led to spontaneous [14C]glutamate release in synaptosomes, which was not accompanied by plasma membrane depolarization. Membrane potential was registered by fluorescent dye DiSC3(5). Y27632 induced an increase of acridine orange fluorescence, exercising no influence over fluorescence of FM2-10 dye. These results suggest that Rho-kinase inhibition decreases pH gradient of synaptic vesicles not inducing exocytosis. Dissipation of the gradient leads to leakage of neurotransmitters to cytosol pumping them out by plasma membrane transporters. Our results show the involvement of Rho-dependent branch of intracellular signaling in regulation of pH gradient in synaptic vesicles.     

Characteristics of Growth Inhibition of Lactobacillus casei by 4-Nitroquinoline-n-Oxide

The bacteriostatic action of 4-nitroquinoline-n-oxide (4-NQO) for Lactobacillus casei is substantially reversed by d-and l-cysteine, glutathione, and 2,2-dihydroxy-1,4-dithiolbutane (dithioerythritol). The action appears to involve a chemical reaction between carbon atom 4 of 4-NQO and nucleophilic centers, such as -SH groups, located on essential cell constituents. The evidence presented indicates that the protective effect of d- and l-cysteine, glutathione, and dithioerythritol against the action involves reactions between 4-NQO and -SH compounds.     

Decreased endo-lysosomal acidification capacity in methylene diphosphonate-resistant mutants of Dictyostelium discoideum

Abstract The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideum amoebae with acridine orange, a fluorescent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 10⁵ and 2.5 × 10⁶ cells ml⁻¹ and independent of acridine orange concentration in the 1.5 to 7.5 μM range. The dye response was sensitive to agents that perturb endo-lysosomal acidification such as NaN₃, nigericin or imidazole. Several mutant cell lines whose growth was resistant to methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lysosomal acidification was altered in these mutants.     

Targeting of Liposomes to Cells Bearing Nerve Growth Factor Receptors Mediated by Biotinylated Nerve Growth Factor

We have used biologically active derivatives of beta-nerve growth factor (NGF), modified by biotinylation via carboxyl groups, to target the specific binding of liposomes to cultured rat and human tumor cells bearing NGF receptors. Liposomes, to be used for targeting, were prepared by conjugating streptavidin to phospholipid amino groups on liposomes prepared by reverse-phase evaporation. Approximately 2,000 streptavidin molecules were incorporated per liposome. Addition of biotinylated NGF, but not of unmodified NGF, could mediate the subsequent binding of radiolabeled streptavidin-liposomes to rat pheochromocytoma PC12 cells in suspension at 4 degrees C. In contrast, incubation with biotinylated NGF did not mediate the binding of hemoglobin-conjugated liposomes. Under optimal incubation conditions, approximately 570 streptavidin-liposomes were specifically bound per cell. Biotinylated NGF was also used to obtain specific binding of streptavidin-liposomes containing encapsulated fluorescein isothiocyanate-labeled dextran to PC12 cells or human melanoma HS294 cells. When HS294 cells were incubated at 37 degrees C following targeted liposome binding at 4 degrees C, the cell-associated fluorescence appeared to become internalized, displaying a perinuclear pattern of fluorescence similar to that observed when lysosomes were stained with acridine orange. Trypsin treatment abolished cell-associated fluorescence when cells were held at 4 degrees C but did not alter the fluorescence pattern in cells following incubation at 37 degrees C. When liposomes containing carboxyfluorescein, a dye capable of diffusing out of acidic compartments, were targeted to HS294 cells, subsequent incubation at 37 degrees C resulted in diffuse cytoplasmic fluorescence, suggesting that internalized liposomes encounter lysosomal or prelysosomal organelles.     

Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.