Exogenous cytochrome c-dependent light-induced membrane potential generation in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores associated with a planar phospholipid macromembrane by bivalent cations in the presence of quinone, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate generate a transmembrane electrical potential difference in the light. Photoelectrical activity is also observed if chromatophores are preincubated with cytochrome c; the maximum values of responses are reached upon subsequent addition of ascorbate and menadion in the absence of bivalent cations and TMPD. The cytochrome c-dependent responses of the illuminated chromatophores are inhibited by Ca²⁺ and prevented by quinones. The possibility of cytochrome c (c₂) translocation across the chromatophore membrane and the mechanism of charge transfer across the planar phospholipid membrane are discussed.     

A permeability transition in liposomes induced by the formation of Ca/palmitic acid complexes

Formation of palmitic acid/Ca²⁺ (PA/Ca²⁺) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca²⁺ showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca²⁺, which was much higher than that of other FFA and lipids. The formation of FFA/Ca²⁺ complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca²⁺ to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca²⁺. The pH-optimum of this phenomenon, similar to that of PA/Ca²⁺ complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca²⁺, some other bivalent cations (Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺) also induce SRB release upon binding to PA-containing liposomes, while Mg²⁺ turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca²⁺ or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca²⁺-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

Fructose bisphosphatase of cestodes of <Emphasis Type="Italic">Bothriocephalus scorpii</Emphasis>

There have been studied the activity and properties of fructose bisphosphatase (FBPase) of Bothriocephalus scorpii parasitizing in pyloric appendages of the goby Myoxocephalus brandti. All subcellular fractions of B. scorpii (12 g/cytosol, 105 g/cytosol, mitochondria, and microsomes) have the FBPase activity. The enzyme has a high affinity to substrate and needs the presence of bivalent cations (Mg²⁺ or Mn²⁺). AMP inhibits the enzyme significantly. Action of various effectors has been studied. The monovalent (Na⁺, K⁺, Li⁺, NH₄ ⁺) and bivalent cations (Zn²⁺ and Cu²⁺) inhibit the enzyme activity.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Optical Anisotropy of Chromatin. Flow Linear Dichroism and Electric Dichroism Studies

Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg²⁺ cations, in contrast to Na⁺ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.     

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing ³¹P-NMR techniques. The addition of Ca²⁺ results in the observation of a “rigid lattice” ³¹P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T₁. This corresponds to a strong and specific headgroup immobilization by Ca²⁺, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H_II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Cationic inhibition of Ca current and Ca-dependent ciliary responses in Paramecium

The Paramecium cell membrane was voltage-clamped under K current suppression conditions. Ciliary beating was registered using high-speed video microscopy. Depolarizing step pulses activated a transient inward current and induced reversed ciliary beating. Very strong positive steps inhibited ciliary reversal during the pulse suggesting inhibition of the Ca influx. We call the potential, which is sufficiently positive to induce transition from reversed to normal ciliary beating, the transition potential. The transition potential rose with increasing external Ca²⁺ showing saturation beyond 1 mM Ca²⁺. Addition of Mg²⁺, Ba²⁺ or K⁺ to the 1 mM Ca²⁺ bathing solution depressed the transition potential in a concentration-dependent manner. The depolarization-activated inward Ca current increased with rising external Ca concentration, and addition of either Mg²⁺, Ba²⁺ or K²⁺ diminished the inward Ca current. The diverging results of Ca²⁺-dependent positive shifts, and Mg²⁺-(Ba²⁺-, K⁺-) dependent negative shifts in transition potential are compared with shifts of V_Imax. It is concluded that external cations bind competitively — in addition to membrane surface charges — to affinity sites of Ca channel, where they specifically modulate permeation of calcium.     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

Gd<Superscript>3+</Superscript>-chelated lipid accelerates solid-state NMR spectroscopy of seven-transmembrane proteins

Solid-state NMR (SSNMR) is an attractive technique for studying large membrane proteins in membrane-mimetic environments. However, SSNMR experiments often suffer from low efficiency, due to the inherent low sensitivity and the long recycle delays needed to recover the magnetization. Here we demonstrate that the incorporation of a small amount of a Gd³⁺-chelated lipid, Gd³⁺-DMPE-DTPA, into proteoliposomes greatly shortens the spin–lattice relaxation time (¹H-T ₁) of lipid-reconstituted membrane proteins and accelerates the data collection. This effect has been evaluated on a 30 kDa, seven-transmembrane protein, Leptosphaeria rhodopsin. With the Gd³⁺-chelated lipid, we can perform 2D SSNMR experiments 3 times faster than by diamagnetic control. By combining this paramagnetic relaxation-assisted data collection with non-uniform sampling, the 3D experimental times are reduced eightfold with respect to traditional 3D experiments on diamagnetic samples. A comparison between the paramagnetic relaxation enhancement (PRE) effects of Cu²⁺- and Gd³⁺-chelated lipids indicates the much higher relaxivity of the latter. Hence, a tenfold lower concentration is needed for Gd³⁺-chelated lipids to achieve comparable PRE effects to Cu²⁺-chelated lipids. In addition, Gd³⁺-chelated lipids neither alter the protein structures nor induce significant line-width broadening of the protein signals. This work is expected to be beneficial for structural and dynamic studies of large membrane proteins by SSNMR.     

Volume contraction of isolated pea chloroplasts promoted by bivalent cations

1. Chloroplasts isolated from pea seedlings were incubated in sucrose–tris medium reinforced with salts of calcium, magnesium, manganese or iron, at concentrations up to 10mm. 2. Measurements of chloroplast-pellet volume and water content showed that the bivalent cations brought about a contraction in chloroplast volume and a loss of chloroplast water. This was further substantiated by density-gradient centrifugations. 3. Measurements of the light-scattering and apparent fluorescence of chloroplast suspensions confirmed this conclusion and eliminated the possibility of contraction being caused by centrifugal forces. 4. The uptake of ⁴⁵Ca²⁺ was measured and shown to be competitive with diluent Ca²⁺, Mg²⁺ or Mn²⁺ ions, indicating a mechanism of low specificity. 5. The chloroplast contraction was insensitive to light but could be made sensitive by the addition of ferric EDTA. This light-sensitivity was inhibited by added 3-(p-chlorophenyl)-1,1-dimethylurea and so probably involves the Hill reaction. 6. On the basis of these observations it is suggested that the process of contraction does not consume much energy, but that in light-activated contraction a previous step occurs that is conducive to contraction and that is energy-transducing. It is postulated that this step results in a local increase in concentration of bivalent ions, which promotes contraction.     

Effects of calcium and manganese ions on the helix–coil transition of DNA

The thermal denaturation method was employed to study the effect of Ca²⁺ and Mn²⁺ ions on the DNA helix–coil transition parameters at Na⁺ concentrations of 10^?3–10^?1M. At low ion concentrations, thermal stability increases, the melting range passes through a maximum, and the denaturation curves become asymmetric. These changes are quantitatively similar for Mn²⁺ and Ca²⁺ ions. With a further increase in the concentration of bivalent ions, the conformational transition temperatures pass through a maximum, and the melting range first tends to saturation and then rapidly decreases to 1–2°C. The Mn²⁺ concentrations, at which the above effects occur, are an order of magnitude lower than the Ca²⁺ concentrations. Comparison of experimental results and calculation in terms of the ligand theory permitted estimation of binding constants characterizing association between Mn²⁺ and Ca²⁺ ions and bases of native and denatured DNA. We show that, unlike the interaction with phosphates, bivalent ion–DNA base binding is weakly dependent on monovalent ion concentration in the solution.     

Prostaglandin synthetase activity of goat vesicular microsomes: Co-factor requirement and effect of calcium ions

The effects of several co-factors and bivalent cations on the activity of prostaglandin synthetase isolated from goat seminal vesicles were studied. Ca²⁺ appears to play a regulatory role in the biosynthesis of prostaglandin E₂ by goat vesicular microsomes as the normal parabolic time course of synthesis changed to a sigmoid curve in the presence of 4 mM Ca²⁺ and to nearly a hyperbolic pattern when the microsomes were preincubated with the metal ions. The Ca²⁺ modulated reaction showed increased rate of prostaglandin E₂ synthesis only when the period of incubation was extended beyond 30 min. The co-factor requirement of the goat enzyme was similar to that of the bovine and ovine prostaglandin synthetase systems.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore

Inorganic polyphosphate (polyP) is a biopolymer of phosphoanhydride-linked orthophosphate residues. PolyP is involved in multiple cellular processes including mitochondrial metabolism and cell death. We used artificial membranes and isolated mitochondria to investigate the role of the polyP in mitochondrial ion transport and in activation of PTP. Here, we found that polyP can modify ion permeability of de-energised mitochondrial membranes but not artificial membranes. This permeability was selective for Ba²⁺ and Ca²⁺ but not for other monovalent and bivalent cations and can be blocked by inhibitors of the permeability transition pore – cyclosporine A or ADP. Lower concentrations of polyP modulate calcium dependent permeability transition pore opening. Increase in polyP concentrations and elongation chain length of the polymer causes calcium independent swelling in energized conditions. Physiologically relevant concentrations of inorganic polyP can regulate calcium dependent as well calcium independent mitochondrial permeability transition pore opening. This raises the possibility that cytoplasmic polyP can be an important contributor towards regulation of the cell death.     

Isolation and Partial Characterization of DNA Topoisomerase I from the Nucleoids of White Mustard Chloroplasts

DNA topoisomerase was isolated for the first time from nucleoids of white mustard (Sinapis alba L.) chloroplasts. The enzyme had a molecular weight of 70 kDa; was ATP-independent, required the presence of mono- (K⁺) and bivalent (Mg²⁺) cations, and was capable of relaxing both negatively and positively supercoiled DNA. These results suggest that the enzyme isolated belongs to the type IB DNA topoisomerases.