gramicidin-a and 3-3--dipropyl-2-2--thiadicarbocyanine

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dimyristoylphosphatidylcholine; Fluoresceins; Gramicidin; Lipid Bilayers; Membrane Potentials; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Staphylococcus aureus

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

Topics: 1-Naphthylamine; Amino Acid Substitution; Anti-Infective Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane Permeability; Circular Dichroism; Glycine; Gram-Negative Bacteria; Gram-Positive Bacteria; Gramicidin; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Potentials; Microbial Sensitivity Tests; Peptides, Cyclic; Protein Structure, Secondary; Structure-Activity Relationship; Yeasts

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.

Topics: Aminacrine; Anacardic Acids; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Dose-Response Relationship, Drug; Fluorescent Dyes; Gramicidin; Hydrogen-Ion Concentration; Liposomes; Membrane Potentials; Models, Chemical; Molecular Structure; Salicylates; Spectrometry, Fluorescence; Valinomycin

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Benzothiazoles; Carbocyanines; Circular Dichroism; Fluorescent Dyes; Gramicidin; Intercalating Agents; Kinetics; Lipid Bilayers; Membrane Potentials; Protein Conformation; Spectrometry, Fluorescence; Thermodynamics

In human T (Jurkat) lymphoblasts we have studied the calcium signals induced by monoclonal antibodies reacting with the T-cell antigen receptor complex (TCR and CD3). Jurkat cells were preloaded with the fluorescent calcium indicator Indo-1 and the stimulus-induced rise in cytoplasmic free calcium concn was followed in the absence or in the presence of external calcium. The technique allowed the separate investigation of the intracellular calcium release and the external calcium influx processes. The changes in the membrane potential of Jurkat cells were followed simultaneously by using fluorescent indicators. We found that the activation of protein kinase C by phorbol ester (PMA) or by the permeable diacyl glycerol, DiC8, rapidly eliminated the calcium signal, independently of the presence or absence of external calcium, while these treatments did not appreciably change the membrane potential. In contrast, cell membrane depolarization achieved by various treatments selectively blocked the stimulus-induced calcium influx, while did not affect stimulus-induced calcium release from internal stores. The magnitude of the stimulus-induced calcium influx was found to be largely independent of the external calcium concns between about 2-2500 microM. It is demonstrated that the inhibitory effect of membrane depolarization on calcium influx is not simply due to the reduction of the inward calcium gradient under these conditions. These observations indicate a significant down-regulation of the stimulus-induced calcium signal by protein kinase C activation and a selective inhibition of the receptor-operated calcium channels by membrane depolarization.

Topics: 4-Chloromercuribenzenesulfonate; Antibodies, Monoclonal; Benzothiazoles; Biological Transport; Calcium; Carbocyanines; Cell Line; Diglycerides; Gramicidin; Humans; Membrane Potentials; Muromonab-CD3; Protein Kinase C; T-Lymphocytes; Tetradecanoylphorbol Acetate

The role of membrane potential in the activation of human platelets by thrombin, ADP and PAF was assessed, using the fluorescent probe diSC3(5). Thrombin, ADP and PAF transiently depolarised the platelet membrane by 6-8 mV from its resting level (-70 mV). This depolarisation had a similar time course to that of shape change. The ionophores valinomycin and gramicidin hyperpolarised and depolarised the platelets respectively but did not activate them. In contrast, exposure of platelets to high K+ media both depolarised and caused them to change shape. Removal of Na+ from the suspension media abolished the depolarisation induced by thrombin, ADP and PAF but the platelets under these conditions were still capable of changing shape and aggregating. This result indicates that the observed depolarisation depends on Na+ fluxes. Amiloride or tetrodotoxin did not mimic the effect of Na+ removal suggesting that any Na+ movement involved does not go through the classic "Na+ channel". Thrombin, ADP and PAF still depolarised the platelet membrane in the absence of added Ca++. Under these conditions, however, the membrane did not repolarise. It is evident that all three agents, thrombin, ADP and PAF, change the membrane potential of human washed platelets through a similar mechanism and this change seems to be a consequence of stimulus-receptor interaction (and platelet activation?). A causal relationship however between these events cannot be clearly shown.

Topics: Adenosine Diphosphate; Amiloride; Benzothiazoles; Blood Platelets; Carbocyanines; Cell Membrane; Fluorescent Dyes; Gramicidin; Humans; Membrane Potentials; Platelet Activating Factor; Platelet Aggregation; Potassium Chloride; Quinolines; Tetrodotoxin; Thrombin; Valinomycin; Verapamil

The membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the noncovalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-perm-selective membrane. The resting potential calculated from the "valinomycin null point" (the K+ concentration gradient at which valinomycin did not change the potential) was approximately -60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl- permeability, demonstrated by replacing Cl- with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.

Topics: Benzothiazoles; Blood Platelets; Carbocyanines; Cell Membrane Permeability; Egtazic Acid; Fluorescent Dyes; Gramicidin; Humans; Ion Channels; Membrane Potentials; Platelet Aggregation; Potassium; Valinomycin

Topics: Amino Acids; Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Diffusion; Fluorescent Dyes; Glucose; Glutamates; Gramicidin; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney; Microvilli; Rabbits; Sodium; Valinomycin