Impermeant potential-sensitive oxonol dyes: I. Evidence for an “On-off” mechanism

Summary This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membranepotential changes, denoted E _m . Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and E _m . This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes ofE _m . The wavelength dependence of the absorption signal is consistent with an on-off mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.     

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.     

Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

Summary We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from –150 to +150 mV. The data are consistent with the on-off mechanism proposed by E. B. George et al. (J. Membrane Biol.103:245–253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5–10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.ASW, Carnegie-Mellon University     

Identification of phosphate granules occurring in seedling tissue of two palm species (Phoenix dactylifera and Washingtonia filifera)

Haustoria of two palm species, Phoenix dactylifera L. (date) and Washingtonia filifera (Lindl.) Wendl were carefully dissected from seeds, and the ultrastructural characteristics of the large, electron-opaque granules present in the cells of this tissue were compared using standard aldehyde and OsO₄ fixations. In Washingtonia, the granules were smaller than those in date and were more variable in size and presence of non-opaque inclusions. All granules appeared to be membrane bounded although they often filled the bounded space. No protein, lipid, carbohydrate or tannins were found in the granules by standard staining procedures. The granules stained positively with two different metallic-phosphate stains which contained either bismuth or lead. Energy dispersive X-ray microprobe analysis, done on aldehyde-fixed granules and those stained with both phosphate stains, confirmed the fact that phosphorus and calcium were present in the granules. The granules also bound the metallic stains as expected. All procedures consistently confirmed the presence of phosphate in the granules. The data are most consistent with the hypothesis that the granules are composed of polyphosphate.Abbreviations and symbols EDAX energy-dispersive X-ray microanalysis - K K shell peak - K K shell peak - L L shell peak - L L shell peak - M M shell peak     

Cyanine dye structural and voltage-induced variations in photo-voltages of bilayer membranes

Summary Flash illumination alters the voltage across bilayer lipid membranes in the presence of certain cyanine dyes. The waveforms of the photo-voltage vary systematically with dye structure and imposed transmembrane voltage. Experimental results are reported for 27 positively charged cyanine dyes, primarily oxazole derivatives, using lecithin/oxidized cholesterol bilayer membranes and 10-mm sodium chloride solutions. Several dyes do not induce any photo-voltages. Examples are 3,3 diethyl 9 ethyl 2,2 oxacarbocyanine iodide, 3,3 diethyl 2 oxa 2 thiacyanine iodide, and 3,3 dimethyl 2,2 indocarbocyanine iodide. Several dyes, when added to one side of the membranes, induce monophasic waveforms. Examples are 3,3 dimethyl 2,2 oxacarbocyanine chloride, and 3,4,3,4 tetramethyl 2,2 oxazalinocarbocyanine iodide. Other dyes induce a photo-voltage only if transmembrane voltages are imposed. These waveforms are biphasic with some dyes (3,3 diethyl 2,2 oxacarbocyanine iodide, for example) and monophasic with other dyes (3,3 dibutyl 2,2 oxacarbocyanine iodide, for example).The photo-voltage waveforms are explained by models that consider the movement of charged dye molecules within the membrane, following optical excitation. The dye movements are probably induced through charge rearrangements in the dye associated with long-lived triplet states, isomerization, or through excimer formation. These results provide information on the location and orientation of the dye molecules within bilayer membranes. The variations which occur in the waveforms with applied voltage indicate that these membranes are fluid in the direction perpendicular to the membrane plane.     

Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Gonoid thelytoky in soft scale insects (Coccidae: Homoptera)

Cytological analysis of the thelytokous soft-scale insects Coccus hesperidum L. (2n=14) and Saissetia coffeae (Walker) (2n=16) revealed that while in both species the chromosomes did not pair during prophase I, meiosis consisted of two divisions, the chromosome number was reduced, and diploidy was restored by the fusion of the female pronucleus with the polar nucleus II. The difficulty of trying to classify this type of thelytoky as either automictic or apomictic led to the proposal that a new criterion and new terms be used to classify thelytoky (and parthenogenesis). The new criterion is whether the number of chromosome elements present in the first (or only) metaphase of oogenesis is the same as that present in the oogonia (gonoid thelytoky) or different from it (agonoid thelytoky). The new criterion is superior to the existing criteria because it is unambiguous, and because it groups together forms with a similar tendency towards heterozygosity (or homozygosity). The possible evolution of the forms analyzed as well as the two other thelytokous forms of each species described by Thomsen (1927) are discussed. Another soft-scale insect, Physokermes hemicryphus Dalam, consisted of a diploid (2n=18) and a triploid (3n=27) form, in both of which the chromosomes also did not pair. Each of the three species contained a strain in which only a single nucleolus was present per cell. In C. hesperidum some strains with two nucleoli differed in the size of the nucleoli.     

Preparation and characterization of methyl green tetrafluoroborate

Summary Commercial methyl green dyes were converted to tetrafluoroborate by means of NaBF₄-solution, the compounds thus obtained were analytically pure. It was shown to be possible to distinguish between a methyl and an ethyl compound by means of NMR spectroscopy. The dyes are stable in buffered aqueous solution, and in crystalline form. A spectrophotometric assay is proposed.     

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum

Summary The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca²⁺ transport. The absorbance and fluorescence responses of 3,3-diethyl-2,2-thiadicarbocyanine, 3,3-dimethyl-2,2-indodicarbocyanine and oxonol VI during ATP-dependent Ca²⁺ transport are influenced by the effect of accumulated Ca²⁺ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca²⁺ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca²⁺, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulumin vivo andin vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca²⁺ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca²⁺ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca²⁺ translocation. The potential generated during Ca²⁺ transport is rapidly dissipated by passive ion fluxes across the membrane.     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

Indications of a common folding pattern for VDAC channels from all sources

Previous research on the mitochondrial channel VDAC from the yeastS. cerevisiae had identified protein strands forming the wall of VDAC's aqueous pore. Here we report the results of analyzing the primary sequences of VDAC from various sources to see if the transmembrane folding pattern identified from this yeast is conserved for VDAC of different species. We analyzed the primary sequences of VDAC from higher plants, fungi, invertebrates, and vertebrates and found that all have a very similar -partern profile with 12–15 peaks indicating potential sided beta strands that are candidates for protein strands forming the wall of the aqueous pore. All these VDAC sequences can be put into the 13 transmembrane strand folding pattern previously identified for yeast VDAC. These folding patterns agree with available experimental data: both electrophysiological and protease digestion data. Although the primary sequences of VDAC from very diverse organisms show low homology, sequence similarity in the proposed corresponding 13 transmembrane strands is substantial. Competing proposals utilizing 16 transmembrane strands are in conflict with electrophysiological experimental observations and violate the constraints on such strands, such as no charged amino acids facing the phospholipid membrane and sufficient number of residues to span the membrane.     

Structure and physical chemistry of membranes

Summary With the concept of the unit membrane as a central theme, a number of current problems for the theory of membrane structures are discussed from the point of view of the physical chemist. In view of the wide variety of chemical compositions of membranes, some current opinions of the forces maintaining the integrity of membranes are of limited generality. The thermodynamic status of the membrane is discussed, and it is suggested that the new small-systems thermodynamics can be usefully employed as a conceptual basis for discussing fluctuations and phase-changes in membranes. Arguments are given to suggest that the lipid interior of membranes is more ordered and crystalline than is currently supposed. The role of water in membrane structures remains an enigma.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

Electron microscopy of taste buds of the rat

Summary The taste buds of the circumvallate papillae have been examined by electron microscopy in OsO₄-fixed, PTA stained material or after KMnO₄ fixation. The microvilli of the receptor cells have terminal dilatations which presumably give an increased surface area for transduction. The extracellular spaces at the necks of the receptor cells near the bases of the microvilli are interrupted by closed contacts.The synapses have a well defined synaptic cleft suggesting a chemical rather than an electrical mode of transmission. Synaptic membrane specialisations differ from the membrane thickenings of other types of synapse. Presynaptic dense projections are present but there is no well define postsynaptic thickening. Vesicles occur in both pre- and postsynaptic components, but it is debatable whether or not they should be termed synaptic vesicles. Acknowledgements. We are indebted to Professor J. Z. Young, F. R. S., for his stimulating support, and to Mr. S. Waterman for skilled photography.     

Kinetics of the potential-sensitive extrinsic probe oxonol VI in beef heart submitochondrial particles

Summary The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law withk ₂3×10⁵ m ^–1 sec^–1. For the ATP pulse experiments, the slower process follows first-order kinetics withk ₁0.3 sec^–1. In oxygen pulse experiments to an anaerobic dyeparticle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.     

Cell permeability as a rate limiting factor in the microbial reduction of sulfonated azo dyes

Summary Permeabilization of cells of B. cereus and other bacterial strains by toluene treatment significantly increased the passage of sulfonated and carboxylated azo dyes from the external medium into the cells with a concomittant increase of the reduction rate of the dyes. Dyes which are not reduced at all by intact cells were readily decolorized. The reduction rate of sulfonated compounds was consistently larger than of their carboxylated analogues, once the dyes had entered the cells.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.