Interaction of peptides with biomembranes assessed by potential-sensitive fluorescent probes.

Peptide-membrane interaction is an important step to be evaluated in a study of the activity and mode of action of several bioactive peptides. A variety of methods are available; however, few of them satisfy the criteria of being sensitive, biocompatible, versatile, easy to perform, and allowing real-time monitoring as the use of potential-sensitive fluorescent probes. Here we review methods for detecting the effects of membrane-active peptides, even those that are not intrinsically fluorescent, on the different types of membrane potentials, with a special emphasis on studies conducted with living cells. FPE is a probe sensitive to surface potential and detects electrostatic interactions at the water-lipid interface. Di-8-ANEPPS is sensitive to dipole potential and detects membrane incorporations. Transmembrane potential changes reveal major membrane destabilizations, such as in pore formation. The combination of the information obtained from the three potential variations can lead to a more elucidative picture of the mechanisms of the interaction of relevant peptides with biomembranes.     

Membrane studies with polarity-dependant and excimer-forming fluorescent probes

1. The interaction of electron-transporting particles from heavy mitochondria of ox heart with several fluorescent probes was examined. 2. 1-Anilinonaphthalene-8-sulphonate and 2-(N-methylanilino)naphthalene-6-sulphonate both show an energy-dependent response. 3. Energy transfer between the electron-transporting particles and the dyes and the kinetics of the dye-particle interaction were studied in order to locate the binding regions in the membrane. 4. The energy-dependent probe responses were shown to be a result of changes in the quantum yield of fluorescence of the bound dyes together with increased binding of the dyes to the energized membrane. 5. Fluorescence lifetime measurements were also used to observe changes on energization. 6. A new type of probe was found in pyrene-3-sulphonate, which may be regarded as a ;volume indicator' for the internal membrane binding region, since it shows a concentration-dependent excimer fluorescence. 7. By comparing the responses of all these dyes when energized particles are uncoupled, a membrane transition with a time-constant of 2-3s is inferred.     

Further studies of nerve membranes labeled with fluorescent probes

Summary By using the technique of intracellular perfusion combined with fluorescence measurements, the mode of binding of 6-p-toluidinylnaphthalene-2-sulfonate (2–6 TNS) in a squid giant axon was examined. The apparent dissociation constant for the binding sites in axons was found to be roughly 0.22mm. Out of approximately 5×10¹⁴ molecules/cm² of 2–6 TNS bound to the sites in and near the axonal membrane, roughly 2×10¹⁰ molecules/cm² are shown to contribute to a transient decrease in fluorescence during nerve excitation. By recording fluorescence signals with a polarizer and analyzer inserted in four different combinations of orientations, studies were made of the directions of the transition moments of various probe molecules relative to the longitudinal axis of the axon. Among hydrophobic probes examined, the polarization characteristics of the fluorescence signals obtained with 1–8 derivatives of aminonaphthalenesulfonate (1-8 ANS, 1-8 TNS and 1-8 AmNS) were found to be very different from those obtained with 2–6 derivatives (2-6 ANS, 2-6 TNS and 2-6 MANS). A tentative interpretation is proposed to account for this difference in physiological behavior between 1–8 and 2–6 derivatives. It is emphasized that measurements of fluorescence polarization yield significant information concerning the structure of the axonal membrane.     

Lipid-specific fluorescent probes in studies of biological membranes

Lipid-specific fluorescent probes are natural lipids carrying an apolar fluorophore in one of the hydrocarbon chains. Since such probes retain the head groups and resemble the molecular shape of native membrane lipids, they largely mimic the behaviour of their natural prototypes in biological membranes. Information provided by the lipid-specific probes is more differentiated and easier to interpret than that obtained from non-lipid probes. The principles of design of lipid-specific probes are formulated and the relative advantages and disadvantages of various fluorophores are discussed. In order to reduce ambiguities caused by perturbation of the probe environment, it is proposed to use, in a comparative manner, two or more lipid-specific probes resembling each other in all aspects except the polar head groups (the 'two probes' concept). Two types of fluorophores, the anthrylvinyl group and the perylenoyl group, were found to be well suited for the synthesis of lipid-specific probes. Use of both types of probes 'in tandem' opens new possibilities for studying lipid-protein and lipid-lipid interactions in biological membranes. The anthrylvinyl- and perylenoyl-labeled lipids were applied in studies of serum lipoproteins and erythrocyte membranes. A new highly sensitive ligand-receptor binding assay and a new approach to biological signal amplifying based on the use of lipid-specific probes are described.     

Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.     

Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.     

A new approach to studies of cell-antibody interactions using fluorescent lipid probes

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.     

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.     

Interaction of cyanine dyes with nucleic acids. XII.beta-substituted carbocyanines as possible fluorescent probes for nucleic acids detection.

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.     

Interaction of propranolol with model phospholipid membranes. Monolayer, spin label and fluorescent spectroscopy studies

The interaction of propranolol with model phospholipid membranes was studied using various experimental techniques. The partition coefficient of propranolol in the negatively charged membranes of vesicles prepared from phosphatidylserine and phosphatidic acid was found to be more than 20-times higher than in neutral phosphatidylcholine membranes. Preferential interaction of propranolol with acidic phospholipid membranes was confirmed using the monolayer compression isotherm technique and the spin-labelling method. Phosphatidylserine monolayers were markedly expanded even at a relatively low drug concentration (5 . 10(-6) M). In contrast, the effect of propranolol on phosphatidylcholine monolayers was much smaller, being detectable only at a higher concentration of the drug (1 . 10(-4) M). Spin-labeling experiments show that propranolol exerts marked ordering effect on bilayers prepared from acidic phospholipids and does not change the order parameter of phosphatidylcholine membranes. The dependence of the propranolol fluorescence spectrum on the polarity of the solvent allowed us to identify the intercalation region of the drug in the membrane. The fluorophore moiety of propranolol was found to be localized in the lipid polar head groups region of the bilayer. The role of electrostatic and hydrophobic effects in propranolol-membrane interaction is discussed and the effect of propranolol on the ordering of phospholipid bilayers is compared with the effects of other anesthetic-like molecules.     

Interaction of modified cyclodextrins with cytochrome P-450

The effects of modified cyclodextrins (CDs) hydroxypropyl-beta-CD and methyl-beta-CD were studied in vitro on cDNA-expressed human cytochrome P-450 (CYP) activities (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). The modified CDs inhibited the activities of CYP2C19 and CYP3A4 while enhancing CYP2C9 activity by 140 to 176% relative to the control values at lower concentrations. In addition, methyl-beta-CD inhibited CYP1A2 and CYP2D6 at higher concentrations.     

绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

Radioimmunoassay for PMSG and its application to in-vivo studies.

A double-antibody radioimmunoassay for PMSG, especially for meauring PMSG in cattle blood after exogenous application, has been developed. A rabbit antiserum against PMSG and pure PMSG for radioiodination were used. There was a strong cross-reaction against equine LH and FSH, but the slight cross-reaction against bovine LH and FSH could be eliminated by adding bovine LH to each tube during the assay. Unspecific, interfering influences of equine or cow serum could be eliminated by adding a constant amount of PMSG-free serum to each tube. PMSG added to 200 microliter of serum could be recovered by this method with a mean of 90 . 5 +/- 9 . 9%. Inhibition curves obtained with pregnant mare serum or cow serum after administration of PMSG were parallel to those obtained with the PMSG standard preparation. The intra-assay coefficient of variation (CV) was 6 . 9%. The inter-assay CV was 12 . 6%. Sensitivity of the assay was 1 mi.u. PMSG/tube. Values of PMSG measured in the serum of pregnant mares by this assay were comparable with those obtained by a bioassay on the same samples. PMSG was still measurable in blood serum about 10 days after injection of 1500-3000 i.u. PMSG. After infusion of 12,000 i.u. PMSG for 3 h (2 heifers), the half-life of PMSG was found to have two components, one of 51 or 40 h and a slower one of 123 or 118 h.