Fluorescent probes of membrane surface properties.

We have studied the properties of two new fluorescent probes, 7-dimethylaminocoumarin derivatives, 4-[N, N-dimethyl-N-(n-tetradecyl)ammoniummethyl]-7-(N,N-dimethylamino)co umarin chloride (TAMAC) and 4-(n-dodecylthiomethyl)-7-(N,N-dimethylamino)coumarin (DTMAC) in model membrane systems. Both probes are sensitive to solvent polarity. The TAMAC probe has a quaternary ammonium function to position it at a fixed location with respect to the membrane interface. In membranes of dipalmitoleoylphosphatidylethanolamine (DiPoPE), both probes detect marked increases in surface hydrophobicity as the bilayer to hexagonal phase transition temperature is approached. This does not occur when the probes are embedded in dipalmitoleoylphosphatidylcholine (DiPoPC) in which case the fluorescence emission is found to be largely independent of temperature. A nitroxide quencher covalently linked to the 5 position of the sn-2 acyl chain of phosphatidylcholine quenches the fluorescence of DTMAC in DiPoPC more than in DiPoPE, indicating the deeper insertion of this probe in DiPoPC. As the temperature is increased the DTMAC fluorophore moves even further out of the membrane. These findings indicate that DTMAC, which does not contain a group to fix its location along the bilayer normal, adjusts its position to small changes in environment polarity, so as to maintain an environment of a fixed dielectric constant. However, with greater changes in membrane interfacial polarity the environment of the probe will be altered. Thus, in addition to the sensitivity of these probes to solvent polarity, the ability of a fixed nitroxide to quench DTMAC becomes another parameter with which to characterize membrane properties with these probes.

Monovalent cations differentially affect membrane surface properties and membrane curvature, as revealed by fluorescent probes and dynamic light scattering.

The effects of monovalent cations on the interfacial electrostatic potential (psi d), hydrodynamic shear boundary distance (ds), and membrane curvature were studied in large unilamellar phospholipid and galacto/sulfolipid liposomes containing different fractions of negatively charged lipids. The differential effects of alkali metal ions on psi d could be accurately determined at physiological surface charge densities with a surface-anchored fluorescent probe. Li+ and Na+ more effectively decrease psi d and exhibit higher association constants (Kas) than K+ and Cs+. These two groups of cations display qualitatively different perturbations of the interfacial structure. Combining Kas values with the electrokinetic (zeta) potentials yielded the respective ds values. At low ionic strength ds more substantially increases with Li+ or Na+ than with K+ or Cs+. Increasing surface charge density causes increased membrane curvature in the presence of K+ or Cs+, but this is largely prevented by Li+ or Na+. Membrane binding of the amphiphilic cation acridine orange decreases surface charge and membrane curvature more extensively than H3O+, Li+, and Na+. The differential interface-perturbing behavior of monovalent cations is discussed with regard to their different hydration tendencies that will modulate the extent and stability of the hydrogen-bond network along the charged membrane surface.

Probing biomembrane interfacial potential and pH profiles with a new type of float-like fluorophores positioned at varying distance from the membrane surface.

Fluorophores of a new type were synthesized to probe the electrostatic potential or pH profiles in the external interface of biomembranes. The probes consist of the pH-sensitive fluorophore 7-hydroxycoumarin, coupled to a tetradecyl (myristyl) tail by a spacer group of varying length. A positively charged group is included between the tetradecyl and spacer groups to encourage a float-like alignment in the membrane head-group region. Three probes of this type were compared with 4-heptadecyl-7-hydroxycoumarin the fluorophore of which is embedded in the lipid head-group domain. Thus, a ruler-type positioning of the fluorophores was obtained at about 0.2, 0.6, 1.0, and 1.3 nm from the surface. The membrane-bound probes were tested in well-defined liposomes prepared by extrusion with different surface charge densities and size. The predicted positioning of the float-like probes is supported by their binding behavior in liposomes and by steady-state and nanosecond time-resolved fluorescence anisotropy, as well as by their accessibility to different quenchers. The interfacial electrostatic potential (psi d) and pH (pHd) values were derived from the observed apparent pKa shifts of the probes. The obtained psi d and pHd profiles as function of the surface potential (psi 0) and distance from the membrane surface are in good harmony with predictions from nonlinear Gouy-Chapman theory. The electrokinetic potentials (zeta) of the liposome series, measured by Doppler-electrophoretic frequency shift of laser light scattering, are in good proportion to the probe data. When bound to yeast cells, these probes monitor interfacial changes in parallel with glucose-induced medium acidification.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction.

Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N'-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with N,N'-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N'-dicyclohexylcarbodiimide, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis[3-propyl-5-oxoisoxazol-4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H(+)-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.

Cellular daunomycin fluorescence in multidrug resistant 2780AD cells and its relation to cellular drug localisation.

Multidrug resistant (MDR) 2780AD human ovarian carcinoma cells were loaded with the fluorescent anticancer agent daunomycin (DN). Fluorescence anisotropy was lower than for corresponding A2780 wild-type cells, indicating that DN was less rigidly bound than in the wild-type cells. Average fluorescence quenching of DN was lower for 2780AD cells. Data were fitted into a model with a highly quenched fraction (fraction A), corresponding to DN intercalated in DNA, and an unquenched fraction (fraction B). The ratio A/B was one order of magnitude lower for the MDR cells than for the wild-type cells. Two other MDR cell lines were investigated and low A/B ratios were found in both cases. Thus, evidence has been provided that in MDR cells the DNA-bound fraction is relatively low and that more free DN is present, for example in acidic vesicles.