Response of an Escherichia coli-Bound Fluorescent Probe to Colicin E1

The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent "all-or-none" nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

Mast cell-lysosomal cationic protein interaction: effects of metabolic inhibitors and decay of activated cells on enhanced fluorescence of anilinonaphthalene sulfonate

It has been shown earlier that the interactions of the isolated rat peritoneal mast cells with cationic protein from rabbit neutrophil lysosomes (band 2 protein) can be studied using anilinonaphthalene sulfonate (ANS) as a fluorescent probe. In the present communication, binding of ANS dye to the mast cells interacting of histamine release by metabolic inhibitors was found to have no effect on enhancement of ANS fluorescence. On the other hand, inhibition of histamine release at high concentration of Ca2+ (14.4 mM) was accompanied by the decrease in enhance fluorescence. In the presence of 7.2 mM of Sr2+, the release of histamine was enhanced with small but significant increase in ANS fluorescence. The cells heated to 42 degrees C partially lost their capacity to release histamine without the loss of enhanced fluorescence. The mast cells interacting with B2 at 10 degrees C for various time intervals showed time-dependent loss in histamine releasing capacity with concomitant loss in enhanced fluorescence. These studies suggest that the enhancement of ANS fluorescence is associated with the early events of the cell membrane caused by interaction of B2 with the cells. The extracellular cations significantly influence this early event.     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

Membrane effects of aminoglycoside antibiotics measured in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate

The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS). Liposomes of PC and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 microM) and high (3 mM) concentrations of calcium. Liposomes containing PIP2 showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PC:PIP2 molar ratios tested. Liposomes containing other anionic phospholipids (PS, PI and PIP) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PC to anionic lipid was increased to 10:1. The aminoglycoside-induced changes of ANS fluorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PIP2 interaction results in phase separation of PC and PIP2 and thus increases the number of available ANS binding sites in PC:PIP2 liposomes.     

Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.     

Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress

The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer’s disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes—regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.     

ANS fluorescence. I. Effect of self-association on the spectral properties of the probe]

The dependence of spectral properties of Mg2+ and NH4+ salt of 8-anilino-1-naphthalenesulfonic acid (Mg-(ANS)2 and NH4-ANS, respectively) on the dye concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. We have shown that the increase in ANS concentrations leads to changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes, observed in aqueous and organic solvents for both salts of ANS, reflect the existence of self-association of the dye molecules. The decrease in fluorescence intensity induced by self-association of the probe molecules is too small to explain a weak fluorescence of ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded ANS molecules upon interaction of this probe with native and molten globule proteins.     

Effect of air ions on L 1210 cells: changes in fluorescence of membrane-bound 1,8-aniline-naphthalene-sulfonate (ANS) after in vitro exposure of cells to air ions

The ability of air ions to produce changes in the electrical properties of L 1210 mouse leukemia cells was tested. The fluorescence of ANS incorporated into a membrane lipid bilayer (measured microphotometrically) was used as a probe. It was shown that the action of air ions of both signs could change (negative ions by increasing and the positive ones by decreasing) the fluorescence intensity of ANS in the cell surface structures or to an imbalance of ions inside and outside the cell. Both possibilities are discussed in the light of the results of experiments using ouabain or biguanide as factors diminishing the intensity of ANS fluorescence.     

Flow cytometric detection of lymphocyte alterations in Huntington's disease

Several recent reports indicate that patients with Huntington's Disease (HD) may manifest membrane abnormalities in a wide variety of cells including peripheral blood lymphocytes. In this study, flow cytometry is used in conjunction with the fluorescent membrane probe, 8-anilino-1-naphthalene sulfonate (ANS), to examine peripheral blood lymphocytes from 16 HD patients and 14 age- and diet-matched control subjects. Increased ANS fluorescence intensity of lymphocytes (p less than 0.02) was found in HD patients as compared to control subjects. These differences are masked when the mean fluorescence of the total leukocyte population is measured, possibly explaining conflicting data of other investigators. These observed differences in ANS fluorescence intensity between HD patients and control subjects support the concept of a gene defect which may be expressed as membrane alterations in non-neural as well as neural cells. The selective alterations of lymphocytes may also reflect altered immunological activity reported in HD.     

Interaction of N-phenyl-1-amino-8-sulfonaphthalene (ANS) with albumin in blood in hyperbilirubinemia

The fluorescent intensity of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe significantly decreases in hyperbilirubinemic serum. A decrease of the albumin concentration and absorption of ANS fluorescence by bilirubin cannot explain such a considerable reduction of the probe fluorescence intensity. Measurements of the fluorescence decay kinetics has shown two types of sites occupied by ANS in albumin. ANS quantum yields in hyperbilirubinemic and normal serum are practically identical. The coupling parameters for ANS decrease, but the coupling constant increases under hyperbilirubinemia. As a result the coupling of organic anions with serum albumin significantly decreases if there is high anion concentration, and it does not decrease at low anion concentration. Bilirubin is not a main cause of a decrease of the albumin binding capacity.     

Interaction of the bacteriocin pediocin SJ-1 with the cytoplasmic membrane of sensitive bacterial cells as detected by ANS fluorescence

The l-anilino-8-naphthalenesulphonic acid (ANS) fluorescent probe was used to monitor alterations in the cytoplasmic membrane of sensitive Lactobacillus plantarurm cells, caused by pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. The addition of pediocin SJ-1 to the sensitive cells caused an increase in fluorescence intensity of ANS and a blue shift in its emission maximum from 520 to 475 nm. None of these spectral changes could be detected when pediocin SJ-1 was added to cells of a Lact. plantarum variant resistant to pediocin SJ-1. Upon the addition of pediocin SJ-1, dose-dependent energy transfer took place between tryptophanyl residues in the cytoplasmic membrane of sensitive cells and ANS. Similar ANS-fluorescence changes were observed with the bacteriocin nisin. The concentrations of pediocin SJ-1 needed to effect changes in the fluorescence spectrum of ANS were of the same magnitude as those required for a bactericidal effect and the release of u.v.-absorbing material. A hypothesis on the mode of action of pediocin SJ-1 is proposed.     

Relationship between ANS fluorescence and electrokinetic potential of activated lymphocytes.

An immune response was induced in vivo on C3H/He ♂ mouse strain with Bovine Serum Albumin (BSA), or Sheep Red Blood Cells (SRBC). The membrane fluorescence changes of activated splenic lymphocytes were studied two weeks after the injection of antigens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on antigens. Their fluorescence intensities were lower than control values. A maximum decrease of fluorescence was recorded on days 1, 6 and 9 after immunization with BSA-stimulated lymphocytes. SRBC-stimulated lymphocytes exhibited a maximum ANS fluorescence decrease on days 4 and 9 after immunization. These fluorescence phenomena would be in an inverse relationship with the electrokinetic surface potential of activated lymphocytes, as assessed by the electrophoretic mobility analysis (EPM). Some parameters affecting the ANS fluorescence in T and B cells are discussed. Quantification of hydrophobic sites in splenic cells would indicate that forces other than the hydrophobic ones may also be involved in the dye-binding changes following immune activation.     

Localization of a 1-anilinonaphthalen-8-sulfonate fluorescent probe on the surface monolayer of low density lipoproteins

A decay of fluorescence probe 1-anilino-8-naphtalene sulfonate (ANS) sorbed on low density lipoproteins (LDL) surface obtained from human plasma was described. It was demonstrated that on the LDL surface the ANS probe is allocated among two pools of molecules with the time of fluorescence decay 4-7 ns and 12-16 ns. One may conclude that 75-90% of the probe is connected with lipid LDL matrix.     

Mechanism of action of small doses of radiation

Opposite changes occur in the intensity of UV-fluorescence (UVF) in irradiated (0.1 Gy and 5.0 Gy) HeLa cells. The radiometric study has demonstrated that there is a correlation between the number of tryptophan-containing proteins and UVF intensity in nonirradiated and irradiated (5.0 Gy) cells during culture growth. Such a correlation was absent in cells exposed to 0.1 Gy radiation. Low radiation doses (0.1 Gy) have maximum action on cytoplasm membrane fluorescence. Low-level radiation changes the intensity of the ANS probe fluorescence connected with cell membranes, and the intensity of the cell protein UVF. High radiation doses increase and low doses decrease the probe fluorescence.     

Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

Intracellular fluorescence decay time of anilinonaphthalene sulfonate

This paper presents the intracellular fluorescence decay time of the probe anilinonaphthalene sulfonic acid (ANS) and compares the results to certain ANS complexes in vitro. There is relatively constant decay time for intracellular ANS over a range of concentrations in the incubating medium, compared with marked variation in results with the complex of ANS-bovine serum albumin in vitro when concentration of the probe is varied. Calculation of the apparent rotational relaxation time from the Perrin equation, using ANS intracellular decay time and polarization data gave a tentative value of circa 66 ns. By comparison with the results of ANS complexes with cell fractions and with certain lipids these data support the concept that intracellularly the compound may be largely membrane located with a portion of the molecule in the lipid phase. Cells damaged by heating or alcohol show longer decay time than those which have taken up ANS in the living state. Suggestions for refinement of technique are included in the discussion.     

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

It is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes. In intact E. coli ML 308-225 cells the inhibition of [¹⁴C]-proline active transport by FCCP increases with uncoupler concentration from ～ 20% at 2 μM to ～100% at 5 μM. The increase in the rotational relaxation time (ρ) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)

1 Abbreviations: FCCP – carbonyl cyanide p-trifluoromethoxyphenylhydrazone; ANS – 8-anilino-1-naphthalenesulfonate; PhNap, N-phenyl-1-naphthylamine; EDTA – ethylenediaminetetraacetate.

and 8-anilino-1-naphthalene-sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in ρ value of ANS show the same dependence on FCCP concentration with saturation at 0.3 μM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5 μM FCCP. It is concluded that (a) EDTA treatment removes a permeability barrier t o FCCP and PhNap in the outer membrane; (b) uncoupling by FCCP removes a similar permeability barrier to PhNap; (c) binding of amphiphilic ANS, assumed to be located in the outer membrane, is hardly changed by these treatments; (d) deenergization of the inner membrane by FCCP thus causes a structural change in the outer membrane as measured by the permeability change to hydrophobic PhNap and the increase in ρ values of the amphiphilic ANS; (e) The binding sites reached by PhNap within the permeability barrier at or near the inner membrane are changed by FCCP from their initial state. This is inferred from an increase in PhNap quantum yield extrapolated to infinite cell concentration, and from removal by FCCP of an apparent phase transition sensed by the PhNap rotational relaxation time. Thus, uncoupling and deenergization by FCCP appears to cause structural change both in the outer membrane and inside the permeability barrier of the outer membrane. Transmission of the colicin E1 response in the envelope of intact and EDTA-treated cells can also be monitored by an increase in ANS and PhNap fluorescence intensity, a smaller fractional increase in dye binding, and a large increase in probe rotational relaxation time. The fluorescence changes of ANS again imply structural effects in the outer membrane caused by colicin. The binding and fluorescence changes of PhNap caused by colicin E1 acting on intact cells again imply an effect of deenergization on the permeability barrier of the outer membrane. Fluorescence changes with PhNap in intact and EDTA-treated cells show that the dye binding sites are altered in the presence of colicin E1. It is also shown that the PhNap intensity change can be blocked by low concentrations of vitamin B12, which competes for the colicin E1 receptor. Some properties are presented of the probe chlorotetracycline, which has been proposed by others to be an indicator of magnesium. The probe appears to reside in an environment somewhat similar to that of ANS, but the colicin-induced changes in its fluorescence parameters appear to be small under our conditions.     

Assessment of conformation changes of immunoglobulin G molecules using a fluorescent probe

A proximate method for conformational changes detection in immunoglobulin G preparations used in research and medical practice is introduced. This method is based on the measurement of IgG-connected 1,8-anilinenaphtalenesulphonate fluorescent probe (ANS) time of damping. When the protein structure is broken fluorescence damping time of IgG-connected ANS rises up.