Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

Interactions involving the cyanine dye,diS-C3-(5), cytochromec and liposomes and their implications for estimations of Δψ in cytochromec oxidase-reconstituted proteoliposomes

Summary The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C₃-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C₃-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide.The cytochromec mediated absorbance and fluorescence losses of diS-C₃-(5) in liposomal or proteoliposomal systems are prevented by Ca²⁺ and La³⁺ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa ₃-reconstituted proteoliposomes is discussed.     

Interactions of cytochromec with phospholipid membranes

Summary Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015m KCl is readily bound. After successive washings with 0.15m KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 170, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10m. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

Some properties of cytochromec' and other hemoproteins ofThiocapsa roseopersicina

Cytochromec' ofThiocapsa roseopersicina was partially purified by DEAE and Sephadex chromatography (highest purity index A 275 nm ox./A 396 nm ox.=0.54). It is autoxidizable, thermostable, and is located in the soluble fraction. The reduced cytochromec' reacts with carbon monoxide and has a γ-band at 417 nm and a shoulder at 435 nm. Cyanide (10⁻²−10⁻³ M) does not inhibit the reduction of the cytochromec' by sulfide; only at higher concentrations of cyanide did the shoulder at 435 nm disappear. When the cytochromec' is reduced by dithionite, only one broad α-band at about 550 nm appears next to the γ-band. When is is reduced by sulfide, the absorption spectrum shows an additional β-band at 521 nm. Cytochromec' is not reduced by thiosulfate.     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Bluelight-induced,flavin-mediated transport of redox equivalents across artificial bilayer membranes

Summary This paper continues our studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochromec, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e ^–(EDTA)-and 2e ^–(BH₃CN^–) donors across the membrane finally reducing the trapped cytochromec both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented.     

Direct monitoring of the electron pool effect of cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>3</Subscript> by highly sensitive EQCM measurements

Cytochrome c₃ from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c₃ with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c₃ molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c₃ is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c₃ to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c₃ and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c₃ stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c₃ facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c₃ donates the accepted electrons to the viologen with difficulty.     

Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochromec

Summary A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochromec.The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1n KCl their maximum direct current (dc) resistance is 7×10⁸ Ohm cm²±10%; the series capacity at 1kHz is 0.43 F/cm²±3%; the entire thickness, determined by optical reflectivity, is 5.8±0.2 nm.The interaction between oxidized cytochromec and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochromec maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochromec.     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Evidence that Basolateral But Not Apical Membrane Localization of Outwardly Rectifying Depolarization-Induced Cl− Channel in Airway Epithelia

The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl⁻ (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl⁻ current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br⁻≒ I⁻ > Cl⁻. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I _sc ) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl⁻ current is present on the basolateral membrane of airway epithelia. Received: 7 October 1999/Revised: 24 April 2000     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

Quantitative conformational analysis of cytochromec bound to phospholipid vesicles studied by resonance Raman spectroscopy

Resonance Raman spectra have been recorded from ferri-cytochromec bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phasphatidylcholine (DOPG-OPC) (70 : 30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interative fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm⁻¹), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochromec adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or finger-print, region (200 to 800 cm⁻¹), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochromec in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented. The proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration, is found to increase on binding ferri cytochromec to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC<DOPG<DMPG, with a parallel decrease of the proportion of the conformational state I, whose structure is similar to that of the uncomplexed ferri-cytochrome c in solution. Similar conformational changes are found for ferro-cytochromec compared to those obtained with the oxidized species on binding to lipids. The present work is essential for studies which seek to analyze, in any detailed fashion, the conformational transitions in the heme protein which take place in response to changes in the lipid environment.     

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells

2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Volume-dependent Glutamate Permeation Depends on Transmembrane Ionic Strength and Extracellular Cl<Superscript>−</Superscript>

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl^– permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl^– on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl^– was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 mM) or low (50 mM) ionic strength solutions on both sides of the membrane, Cl^– was always more permeable than Glu. When a transmembrane ionic strength gradient (150 mM extracellular: 50 mM intracellular) was set to drive water into the cell, and in the presence of extracellular Cl^–, Glu became up to 16-fold more permeable than Cl^–. Replacement of extracellular Cl^– with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.