Photodynamic effect of chlorine e6 on erythrocyte membranes

Photodestruction of erythrocyte membranes sensitized by water soluble chlorophyll derivative chlorine e6 (Chl e6) was studied. It has been determined that light irradiation of erythrocyte ghosts with wave length lambda-660 nm in the presence of Chl e6 caused in protein and lipid components of the membrane deep destructive changes which were expressed in cross-linking of the membrane polypeptides and accumulating in the membrane products of peroxidation of unsaturated fatty acids residues, in phospholipids mainly. It has been shown that these processes were realized only in the presence of molecular oxygen and consequently possess pure photodynamic character.     

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.     

On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

Properties of liposomes that contain chloroplast pigments: Photosensitivity and efficiency of energy conversion

Liposomes that contain chlorophyll and carotene are photosensitive. If a gradient of redox potential exists across the liposome membrane, illumination causes charge transport. The quantum efficiency of energy conversion in liposomes is about 0.075. It appears that chlorophyll aggregates are present in the liposomes and that these aggregates are involved in energy conversion.     

Topology and Growth of the Intracytoplasmic Membrane System of Rhodopseudomonas spheroides: Protein, Chlorophyll, and Phospholipid Insertion into Steady-State Anaerobic Cells

An equilibrium density gradient centrifugation study involving the separation of "old" and "new" membranes has been developed to determine the manner in which protein, lipid, and chlorophyll are incorporated into growing intracytoplasmic membranes (chromatophores) of Rhodopseudomonas spheroides. Chromatophores derived from cells grown in an H(2)O-medium had a density of 1.175 to 1.180 g/cm(3) and were readily separable from chromatophores having a density of 1.220 to 1.230 isolated from cells grown in a 70% D(2)O-medium. After a shift from "D(2)O-" to "H(2)O"-based media, only hybrid chromatophores derived from a combination of "heavy" (old) and "light" (new) chromatophore material could be detected. The experimentally determined, median density values for the growing intracytoplasmic membrane system followed a theoretically determined profile which was calculated from the density of full "heavy" and full "light" material assuming random, homogeneous incorporation of new material into old membrane. The distribution of the radioactive labels for protein (leucine) and chlorophyll (delta-aminolevulinic acid) were identical and showed a reproducible displacement of the "old" material to the heavy side of the optical density at 365 nm (OD(365)) absorbance and a displacement of the "new" material to the light side of the OD(365) absorbance profile. Specific phospholipid growth showed no displacement for either the "old" or "new" material from the median absorbance profile.     

Pigment Degradation in Discs of the Thermophilic Cucumis sativus as Affected by Light, Temperature, Sugar Application and Inhibitors

Chlorophyll degradation in Cucumis leaf discs was measured at different temperatures between 1 and 25°C in the light and in darkness, and in the presence or absence of sucrose. Two different processes of chlorophyll degradation could be distinguished, a light-requiring process operating at 1 and 5°C and another, light and sucrose enhanced degradation process which was evident at 25°C. Degradation of leaf pigments at low temperatures was of a photo-oxidative nature since there was no degradation in the dark. The chlorophyll a/b ratio was decreased, carotene was degraded at a faster rate than chlorophyll, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and triphenyltetrazolium chloride (TTC) which prevent photo-oxidation, protected against chlorophyll degradation. The light and sucrose enhanced chlorophyll degradation at 25°C was of an enzymatic nature since it occurred in the dark as well as in the light. The chlorophyll a/b ratio was not affected, and carotene and chlorophyll degradation occurred at the same rate. Since DCMU completely inhibited the light enhancement at 25°C and experimentation in a low oxygen atmosphere also protected chlorophyll against the effect of light and sugar application, it is suggested that the enhancement of chlorophyll degradation by light and sucrose at 25°C may be due to increased sugar uptake of the chloroplasts and consequently excessive starch formation in the organelles.     

Spin-label studies on rat liver and heart plasma membranes: do probe-probe interactions interfere with the measurement of membrane properties?

The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase separation was observed in liver membranes between 19 degrees and 28 degrees. Addition of CaCl2 to liver plasma membrane [labelled with "low" I(12,3) concentrations] increased the rigidity of the membrane at 31 degrees and 37 degrees, without inducing a segregation of the probe in the bilayer. Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.     

Photodynamic activation of ion transport through lipid membranes and its correlation with an increased dielectric constant of the membrane

Illumination of biological membranes with visible light in the presence of membrane-active sensitizers (e.g. rose bengal) is known to inactivate transport proteins such as ion channels and ion pumps. In some cases, however, illumination gives rise to an activation of transport. This is shown here for ion channels formed by alamethicin in lipid membranes, and for porin channels, which were isolated from the outer membrane of E. coli (OmpC) and from the outer membrane of mitochondria (VDAC) and were reconstituted in lipid membranes. An activation (in the form of an increased conductance) was also observed in the presence of the cation carriers valinomycin and nonactin. The activation phenomena were only present, if the membranes were made from lipids containing unsaturated double bonds. Activation was reduced in the presence of the antioxidant vitamin E.We suggest that the activation of the different transport systems has a common physical basis, namely an increase of the dielectric constant, epsilon(m), of the membrane interior by the presence of polar oxidation products of photodynamically induced lipid peroxidation. Experimental evidence for an enhanced dielectric constant was obtained from the finding of a light-induced increase of the membrane capacitance in the presence of rose bengal.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.     

Determination of chlorophyll porphyrin ring orientation in black lipid membranes by photovoltage spectroscopy

Photovoltage spectroscopy with polarized light has been used to investigate the structure of black lipid membranes formed from spinach chloroplast extracts. The photovoltage action spectrum of the black lipid membranes is similar to the absorption spectrum of the membrane-forming solution, with a red and principal blue peak. The magnitudes of these peaks have been found to depend on the direction of polarization of the exciting light. This is apparently a direct consequence of the dichroism of the membrane. The polarized light photovoltage data have been used to obtain information on the orientation of chlorophyll in the membrane.     

The greening of chromoplasts in Daucus carota L.

Summary Evidence was obtained for the transformation of chromoplasts to chloroplasts in the cortex parenchyma of carrot during exposure to light. Typical chromoplasts containing carotene crystals but no lamellar system were observed at the onset of illumination. The ensuing synthesis of chlorophyll and a lamellar system was accompanied by disappearance of the carotene crystals. Only chloroplasts were present after 48 hr in the light. The different stages of plastid development were observed using the electron microscope.     

Purification of the bifunctional enzyme, imidazoleglycerolphosphate dehydratase-histidinol phosphatase, of Salmonella typhimurium

Chloroplasts isolated from fully developed spinach leaves and incubated in the presence of isopentenyl pyrophosphate were able to synthesize rapidly geranylgeranyl chlorophyll alpha and geranylgeraniol. The biosynthesis of the geranylgeraniol derivatives from isopentenyl pyrophosphate is a compartimentalized process. The membrane fractions (thylakoid and envelope membranes) were essentially unable to synthesize geranylgeraniol, geranylgeranyl pyrophosphate and geranylgeranyl chlorophyll alpha. When stromal and thylakoid fractions were combined the capacity to synthesize geranylgeranyl chlorophyll alpha and geranylgeraniol was restored. When stromal and envelope membrane fractions were combined the capacity to synthesize gernylgeranyl pyrophosphate and geranylgeraniol was restored. The products of the reaction were discharged inside the lipid phase of the membranes.     

Insertion of short amino-functionalized single-walled carbon nanotubes into phospholipid bilayer occurs by passive diffusion

Carbon nanotubes have been proposed to be efficient nanovectors able to deliver genetic or therapeutic cargo into living cells. However, a direct evidence of the molecular mechanism of their translocation across cell membranes is still needed. Here, we report on an extensive computational study of short (5 nm length) pristine and functionalized single-walled carbon nanotubes uptake by phospholipid bilayer models using all-atom molecular dynamics simulations. Our data support the hypothesis of a direct translocation of the nanotubes through the phospholipid membrane. We find that insertion of neat nanotubes within the bilayer is a "nanoneedle" like process, which can often be divided in three consecutive steps: landing and floating, penetration of the lipid headgroup area and finally sliding into the membrane core. The presence of functional groups at moderate concentrations does not modify the overall scheme of diffusion mechanism, provided that their deprotonated state favors translocation through the lipid bilayer.     

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C₄TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C₄TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C₄TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C₄TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C₄TPP-X series, with the exception of C₄TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.     

Fluidizing effects of centrophenoxine in vitro on brain and liver membranes from different age groups of mice

This study examined the effects of different concentrations of centrophenoxine on physical properties of synaptic plasma membranes and liver microsomes using electron spin resonance procedures. Membranes of different age groups of mice were labeled with the 5-doxyl stearic acid spin-label and membrane fluidity determined in the presence and absence of different concentrations of centrophenoxine. Centrophenoxine had a direct effect on membranes as shown by a significant increase in membrane fluidity. This effect was greatest in liver microsomes as compared to synaptic plasma membranes. Age differences were not observed in centrophenoxine-induced fluidization. Effects of centrophenoxine in vivo may be due in part to the drug acting directly on the physical properties of the membrane lipid environment.     

The susceptibility of membrane vesicles to interaction with ethanol

The susceptibility of membranes to interaction with ethanol is an important consideration in the further understanding of the ethanol-membrane interaction. Interaction of membrane vesicles, including passive diffusion of ethanol across membranes, leakage of internal molecules out of membranes and membrane-membrane interaction, were examined systematically using two populations of fluorescent probe-encapsulated phospholipid bilayer vesicles, each prepared with 1,2-dimyristoyl phosphatidylcholine, cholesterol and a fluorescent probe. Fluorescence quenching experiments with these vesicles were performed in a medium containing a wide range of ethanol concentrations (0.30-3.5 M). In the presence of a lower concentration of ethanol in the external medium, passive diffusion of ethanol across membrane vesicles occurred. This was demonstrated by an interaction of ethanol with the encapsulated fluorescence probe molecules inside the vesicles, resulting in an increase in the fluorescence intensity and a shift of the fluorescence emission spectrum to a shorter wavelength. While, in the presence of a higher concentration of ethanol in the external medium, a strong perturbation of lipid bilayers by ethanol was found, leading to an over expansion of membranes and consequently causing the membrane leakage. As a result of this, the initially encapsulated probe molecules leaked out of the vesicles so as to interact with the other probe molecules in the external medium. Consequently, fluorescence quenching was observed. Moreover, studies of the mixture of two populations of fluorescence probe-encapsulated membrane vesicles revealed that ethanol acted on individual membranes and did not promote membrane-membrane interactions. The implication of the present results to the alcohol-mediated expansion of membranes is discussed.     

Early effect of sodium pentachlorophenate on photosynthetic activity of the alga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> Chick. S-39

Early effects of 0.0001–10 mM sodium pentachlorophenate (PCP-Na) on the green alga Chlorella pyrenoidosa Chick. S-39 involves a rapid (within 1–2 min) decrease in the light-induced oxygen evolution by algal cells. The suppressed relative yield of variable chlorophyll fluorescence in C. pyrenoidosa in the presence of high PCP-Na concentrations and its dynamics provide evidence for rapid inactivation of photosystem 2, which is not observed at low concentrations of the toxicant. An analysis of the induction curve of delayed chlorophyll fluorescence in algal cells suggests that PCP-Na at low concentrations disturbs the coupling of electron transport and phosphorylation, whereas at high concentrations it inhibits electron transport and decreases the energy potential of photosynthetic membranes. The early toxic effect of PCP-Na is responsible for subsequent impairment of C. pyrenoidosa productivity.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.