Ionic changes in the mitotic apparatus at the metaphase/anaphase transition

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane- associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.     

Reactions of fluorescent probes with normal and chemically modified myelin.

The fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind to highly purified myelin membranes obtained from bovine brain white matter. Binding of the dyes was markedly increased by environmental conditions which reduce the negative surface potential of the membrane, i.e., cations (La-3+ is greater than Ca-2+ is greater than Na-+,K-+), H-+, local anesthetics, and the antibiotic polymyxin B. Chemical alteration of accessible membrane charged groups affected dye binding in a manner consistent with the hypothesis that such binding is primarily dependent upon the membrane surface potential. Thus, binding was increased by blocking of carboxyl groups via carbodiimide activation and subsequent coupling with neutral amino acid esters, and even more so with a basic amino acid ester (e.g., arginine methyl ester). Dye binding was reduced by succinylation of amino groups, and by hydrolysis of choline and ethanolamine head groups of phospho- and sphingolipids by phospholipase C. Phospholipase C treatment of myelin, or sphingomyelin vesicles, reduced or abolished the augmentation of ANS and TNS binding due to cations, local anesthetics, or polymyxin B. Energy transfer from myelin tryptophan residues to bound ANS occurs, but with low efficiency. Oxidation of membrane tryptophan residues with N-bromosuccinimide, or alkylation with 2-hydroxy (or methoxy)-5-nitrobenzyl bromide, markedly reduced intrinsic membrane fluorescence and energy transfer to bound ANS, but did not significantly affect dye binding or the quantum yield of ANS fluorescence when excitation was at 380nm. Proteolytic digestion removed 6-30% of myelin protein, depending upon the enzyme used, but had no effect on fluorescent dye binding. It is concluded that the binding of the anionic fluorescent probes ANS and TNS to myelin is primarily a function of the membrane surface charge density and net surface potential, as is the case with other biological membranes. Conclusions about the degree of dye binding to membrane lipids or membrane proteins cannot be drawn unless additional studies are carried out on isolated water soluble membrane proteins.     

Use of the fluorescent probe method to study the interaction of barbiturates with biological membranes]

The binding of the negatively charged fluorescence dye ANS and neutral dye NPN2 with lipid and erythrocyte membranes in the presence of barbiturates was studied. It was found that barbiturates decreased the amount of binding sites of ANS and NPN2 with membranes did not affect the quantum yield and the dissociation of the membrane-dye complex. It was shown that all barbiturates investigated were bound with the membranes in a neutral form.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.     

Effects of a series of alcohols on the binding of a fluorescent dye to erythrocyte membranes.

1. The effects of a series of aliphatic alcohols (methanol to octanol) on membrane proteins of erythrocytes were studied by monitoring the flueorescence of a dye (1-anilino-8-naphthalenesulfonic acid (ANS)) that adsorbs to erythrocyte ghost membranes. Low concentrations of all the alcohols reduced the ANS fluorescence of the membrane-ANS suspensions; lent to those which protect against hypotonic hemolysis on intact erythrocytes; higher concentrations markedly increased the fluorescence. Ethanol and methanol decreased ANS fluorescence at all concentrations. 2. Lytic concentrations of saponin did not increase ANS fluorescence and did not modify the membrane action of the alcohols. 3. None of these effects were observed in liposomes prepared from lipid extracts of the erythrocyte membrane. 4. Since the apparent dissociation constant for the ANS-membrane interaction was unchanged in the presence of the alcohols, it was assumed that the fluorescence changes anesthetic concentration of the alcohols alter the conformation of membrane proteins, as indicated by the decreased number of ANS binding sites.     

pH probes respond to redox changes in cytochrome o

N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids. We have shown (Sedgwick, E. G., and Bragg, P.D. (1988) FEBS Lett. 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles. We now show that the pH probes pyranine and quinacrine behave similarly to NPN. The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium. It is insensitive to uncouplers. The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid. The NPN response requires added phospholipid. Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome. This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.     

A study of the permeation of dihexadecyl phosphate vesicles by various anesthetics

A simple spectroscopic method for the evaluation of the effect that perturbers may have on a membrane model is described. The model was made from dihexadecyl phosphate (DHP) bilayers. The perturbers used were unconventional anesthetics (n-alcohols C1-C8; n-hexane and n-pentane) and conventional anesthetics (chloroform, methoxyflurane, halothane and enflurane). The results show a correlation between vesicle permeation by anesthetics and their clinical potency. Two modes of perturbation by which the anesthetics may induce vesicle permeation are proposed.     

Fluorometric estimation of surface potential change associated with chemotactic stimulation in Tetrahymena pyriformis

For the quantitative estimation of surface potential change in intact cells a method was devised with the use of fluorescent probes, 8-anilino-1-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPN). Estimated values in liposomes were compared with changes in the zeta potential determined from electrophoresis. Both values agreed within the experimental variation, showing the usefulness of the method. The method was also applied to Tetrahymena pyriformis, which exhibits chemotaxis to various chemical stimuli. The surface potential change was observed when the cell was stimulated not only by inorganic salts but also by electrically neutral, hydrophobic compounds. The surface potential started to change in accordance with the depolarization of the membrane potential, except for the case of K⁺. Changes in the surface potential of T. pyriformis in response to Ca²⁺ and K⁺ were compared with those in the membrane potential. The quantitative contribution of the surface potential to cell depolarization associated with chemoreception is discussed.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Fluorometric assessment of gram-negative bacterial permeabilization

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.     

The membrane effect of 1-anilino-8-naphthalene sulfonate on transferrin and iron uptake by rabbit reticulocytes

Studies on the effect of 1-anilino-8-naphthalene sulfonate on uptake of transferrin and iron by rabbit reticulocytes show that the inhibitory action of ANS is localized at the membrane level. The intravesicular pH and cellular ATP level were not affected by this anionic probe. ANS shifted the transition temperature and reduced the enthalpy changes of iron uptake by rabbit reticulocytes. These suggested that the drug reduced the membrane fluidity. Hence, ANS disturbed the physicochemical environment of the receptor for transferrin resulting in the perturbation of receptor-mediated endocytosis.     

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

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 tertiary amine local anesthetics on G protein-coupled receptor lateral diffusion and actin cytoskeletal reorganization

Although widely used clinically, the mechanism underlying the action of local anesthetics remains elusive. Direct interaction of anesthetics with membrane proteins and modulation of membrane physical properties by anesthetics are plausible mechanisms proposed, although a combination of these two mechanisms cannot be ruled out. In this context, the role of G protein-coupled receptors (GPCRs) in local anesthetic action is a relatively new area of research. We show here that representative tertiary amine local anesthetics induce a reduction in two-dimensional diffusion coefficient of the serotonin_1A receptor, an important neurotransmitter GPCR. The corresponding change in mobile fraction is varied, with tetracaine exhibiting the maximum reduction in mobile fraction, whereas the change in mobile fraction for other local anesthetics was not appreciable. These results are supported by quantitation of cellular F-actin, using a confocal microscopic approach previously developed by us, which showed that a pronounced increase in F-actin level was induced by tetracaine. These results provide a novel perspective on the action of local anesthetics in terms of GPCR lateral diffusion and actin cytoskeleton reorganization.     

Age characteristics of the interaction between fluorescent probes and thyroxine with the membranes of rat liver microsomes

By means of fluorescent probes, 1-anilino naphthalene-8-sulfonate (ANS), methoxybenzanthrone (MBA) and pyrene, an increase of membrane affinity to ANS, shift of MBA fluorescence maximum to the short-wave spectrum region and a change of the membrane microviscosity were observed in the liver microsome membranes during aging of rats. No significant changes of fluorescent parameters of the probes with rats aging were found in mitochondria. ANS was concurrently displaced by thyroxine, affinity to which significantly increased with aging, and in young animals during hyperthyroidism. The increase of microsome membranes affinity to thyroxine with age is considered as an intracellular mechanism which is involved in the metabolism changes of hypothyroid pattern in rat liver during aging.     

Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate.

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.     

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.