Protective effects of mitochondria-targeted antioxidant SkQ in aqueous and lipid membrane environments.

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels.

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.

Induction of antibiotic production by protease in Bacillus brevis (ATCC 8185).

Bacillus brevis (ATCC 8185) produces an antibiotic peptide, linear gramicidin, in the early stationary growth phase. Since we observed that preculture in milk medium is essential for production of the antibiotic in broth medium, we studied the role of the preculture in the antibiotic production. We found that addition of the supernatant of the precultured milk medium was sufficient to induce gramicidin production in broth medium. Fresh milk medium had no effect. The effector substance in the overnight cultured milk medium was labile at both acidic and alkaline pH and was destroyed by heat. We also found that addition of a protease (for example, bovine pancreas chymotrypsin or Streptomyces griseus protease) instead of the supernatant could induce the gramicidin production. Addition of Mn2+ was not required for the protease-induced production of gramicidin. It is known that B. brevis cells secrete protease into milk medium. But neither use of the protease-pretreated broth medium nor addition of casamino acids to broth medium induced gramicidin production. These results suggest that B. brevis cells secrete a factor for linear gramicidin production, that the inducing factor is protease and that the target of the protease is a substance(s) produced by the bacteria.

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.

[Interaction of gramicidins with the cellular phospholipids of the producer Bacillus brevis]. / Vzaimodeistvie gramitsidina S s fosfolipidami kletok produtsenta Bacillus brevis.

Phospholipid fractions were isolated from the cells of Bacillus brevis var. G.-B. variants, some, of which produced gramicidin S and some did not. As was found by thin layer chromatography, phosphatidyl ethanolamine predominated in the fraction of phospholipids. Interaction of the isolated phospholipids with gramicidin S in vitro resulted in a loss of the antibiotic activity. Presumably, formation of a complex between the polypeptide and phospholipids may decrease the actual concentration of gramicidin S in cells producing the antibiotic.

The blockage of the electrical conductance in a pore-containing membrane by the n-alkanes.

1. In monooelein bilayers made highly conducting by the addition of a fixed amount of o-pyromellitylgramicidin, the membrane conductance has been shown to be strongly dependent on the chain length of the n-alkane with which the membrane is in equilibrium. Thus for n-hexadecane, the conductance is larger by approx. 10(4) times than it is for n-octane. This result is independent of whether the polypeptide is introduced via the aqueous or lipid phases. 2. The observed conductance variations have been accounted for in terms of a mechanism (outlined in earlier publications) which is based on the thickness and tension changes produced in bilayers by the adsorption of n-alkanes. Essentially quantitative agreement between theory and experiment is found.

Ultrastructural, optical and volume changes in brain mitochondria during gramicidin-induced ion uptake.

Gramicidin-induced swelling of rat brain mitochondria was followed spectrophotometrically with a parallel analysis of changes in the ultrastructure, volume and eccentricity ratio of the mitochondria. Under the effect of gramicidin the expansion of the inner compartment occurs with an overall increase in volume. Mitochondria tend to be rounded although not entirely spherical in shape. The reversal of swelling with respiratory inhibitor (KCN, rotenone) results in a shrinkage of the inner compartment and is not accompanied by a proportional reduction of the outer compartment. Mitochondria fail to regain their original shape. It can be concluded that in the course of energy-dependent swelling the ability to contract might be retained, whereas other factors controlling the shape of the mitochondrion are irreversibly affected.