Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.     

Crystal Structure of Cruxrhodopsin-3 from Haloarcula vallismortis

Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.     

Transport energetics of the Cl− pump in Aplysia gut

Basolateral membranes of Aplysia foregut epithelia contain an ATP-dependent Cl⁻ transporter (Cl⁻ pump). Increased activity of the Cl⁻ pump, coupled to apical and basolateral membrane depolarization, changed the Cl⁻ transport energetics across the apical membrane but did not change the vectorially-opposite Cl⁻ transport energetics across the basolateral membrane.     

ATP-dependent chloride transport in plasma membrane vesicles from Aplysia intestine

A Cl⁻-stimulated ATPase activity, which is sensitive to both thiocyanate and vanadate, has been localized to the plasma membrane of Aplysia enterocytes. Utilizing plasma membrane vesicles from Aplysia enterocytes, ATP stimulated Cl⁻ uptake to approximately 2.5-times that of control in a Na⁺, K⁺ and HCO₃⁻-free medium. This ATP-dependent Cl⁻ uptake was sensitive to both thiocyanate and vanadate. These results are consistent with the hypothesis that the active Cl⁻ absorptive process in Aplysia intestine could be a Cl⁻-stimulated ATPase found in the enterocyte plasma membrane.     

Molecular dynamics of water and monovalent‐ions transportation mechanisms of pentameric sarcolipin

The Sarcolipin (SLN) is a transmembrane protein that can form a self‐assembled pentamer. In this work, the homology modeling and all‐atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na⁺, Cl^? and water molecule along the pore channel of penta‐SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na⁺ and Cl^? could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na⁺ and Cl^? could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu²¹ that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na⁺ and Cl^? ion permeability by hydrated and vacuum states. Proteins 2016; 84:73–81. © 2015 Wiley Periodicals, Inc.     

Measurement of intracellular chloride activity in mouse liver slices with microelectrodes

Steady-state membrane potential (V_m) and intracellular Cl⁻ activity (a_Clⁱ) were measured with double-barreled Cl⁻-selective microelectrodes in mouse liver slices. In bathing solutions (33.8° C) containing pyruvate, glutamate, fumarate, and glucose, V_m and a_Clⁱ were −27.6 ± 1.0 mV and 32.6 ± 1.5 mM, respectively. This apparent value of a_Clⁱ exceeded the level required for passive distribution of this ion (a_Cl^eq = 26.4 ± 1.3 mM) by 6.2 ± 1.0 mM. This difference was essentially unchanged in experiments where (i) Na⁺ was replaced by choline, (ii) HCO₃⁻ was removed, and (iii) Cl⁻ was replaced by gluconate. These data argue against the presence of Na⁺- or HCO₃⁻-coupled Cl⁻ transport mechanisms in the plasma membrane of mouse liver cells. This implies that a_Clⁱ is in fact at equilibrium and interference with the response of Cl⁻-selective microelectrodes by intracellular anions is responsible for the apparent difference between a_Clⁱ and a_Cl^eq. We found that Cl⁻-selective microelectrodes containing Corning 477315 ligand are sensitive to taurocholate, a representative bile salt. Their selectivity to taurocholate is about 60-times their selectivity towards Cl⁻. This suggests that interference of bile acids at concentrations normally present in hepatocytes with determinations of a_Clⁱ can account for the apparent difference a_Clⁱ − a_Cl^eq.     

Cl− influx across posterior gills of the Chinese crab (Eriocheir sinensis): potential energization by a V-type H+-ATPase

Cl⁻ absorption across isolated, perfused gills of freshwater adapted Chinese crabs (Eriocheir sinensis) was analysed by measuring transepithelial potential differences (PD_te) and radioactive tracer fluxes across isolated, perfused posterior gills. Applying hemolymph-like NaCl salines on both sides of the epithelium PD_te amounted to −30±1 mV (n=14). Undirectional Cl⁻ influxes of 470±38 and effluxes of 245±27 μmol·hr⁻¹·g⁻¹ wet weight (ww) (n=14) resulted in a Cl⁻ net influx of 226±31 μmol·hr⁻¹·g⁻¹ ww. Symmetrical substitution of Na⁺ by choline resulted in a substantial hyperpolarisation of the gill. Cl⁻ influx was unchanged under these conditions. However, net influx of Cl⁻ decreased by 40%, due to an increase of the Cl⁻ efflux.Nevertheless, a significant Cl⁻ net influx remained which was independent of the presence of Na⁺. When 2 mmol/l ouabain were added to the internal perfusion medium, PD_te increased, although the fluxes remained unchanged. Following external application of 1μmol/l of the V-type H⁺-ATPase inhibitor bafilomycin, A_l PD_te and Cl⁻ effluxes were not significantly affected. However, Cl⁻ influxes decreased. These findings suggest that Cl⁻ can be taken up independently of Na⁺ and that active Na⁺ independent Cl⁻ uptake across the posterior gill of Eriocheir sinensis is probably driven by a V-type H⁺-ATPase localized in the apical membrane.     

Conformational exchange of fatty acid binding protein induced by protein-nanodisc interactions

Fatty acid binding proteins (FABPs) can facilitate the transfer of long-chain fatty acids between intracellular membranes across considerable distances. The transfer process involves fatty acids, their donor membrane and acceptor membrane, and FABPs, implying that potential protein-membrane interactions exist. Despite intensive studies on FABP-membrane interactions, the interaction mode remains elusive, and the protein-membrane association and dissociation rates are inconsistent. In this study, we used nanodiscs (NDs) as mimetic membranes to investigate FABP-membrane interactions. Our NMR experiments showed that human intestinal FABP interacts weakly with both negatively charged and neutral membranes, but it prefers the negatively charged one. Through simultaneous analysis of NMR relaxation in the rotating-frame (R_1ρ), relaxation dispersion, chemical exchange saturation transfer, and dark-state exchange saturation transfer data, we estimated the affinity of the protein to negatively charged NDs, the dissociation rate, and apparent association rate. We further showed that the protein in the ND-bound state adopts a conformation different from the native structure and the second helix is very likely involved in interactions with NDs. We also found a membrane-induced FABP conformational state that exists only in the presence of NDs. This state is native-like, different from other conformational states in structure, unbound to NDs, and in dynamic equilibrium with the ND-bound state.     

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm⁻¹ structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.     

Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule

Ionic Cl⁻ currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl⁻ conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying I-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I⁻ > Br⁻ > Cl⁻ > > glutamate. The forskolin-activated Cl⁻ conductance demonstrated a linear I-V relationship and its ion selectivity was Br⁻ > Cl⁻ > I⁻ > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl⁻ currents, and DIDS inhibited only the swelling-activated Cl⁻ current. Forskolin had no effect on the activation of the swelling-activated Cl⁻ current. In DCTb cells which exhibited swelling-activated Cl⁻ currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl⁻ currents. In the continuous presence of I⁻ which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl⁻ conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.     

Specific Binding of Chloride Ions to Lipid Vesicles and Implications at?Molecular Scale

Biological membranes composed of lipids and proteins are in contact with electrolytes like aqueous NaCl solutions. Based on molecular dynamics studies it is widely believed that Na⁺ ions specifically bind to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, whereas Cl⁻ ions stay in solution. Here, we present a careful comparison of recent data from electrophoresis and isothermal titration calorimetry experiments as well as molecular dynamics simulations suggesting that in fact both ions show very similar affinities. The corresponding binding constants are 0.44(±0.05) M⁻¹ for Na⁺ and 0.40(±0.04) M⁻¹ for Cl⁻ ions. This is highlighted by our observation that a widely used simulation setup showing asymmetric affinities of Na⁺ and Cl⁻ for POPC bilayers overestimates the effect of NaCl on the electrophoretic mobility of a POPC membrane by an order of magnitude. Implications for previous simulation results on the effect of NaCl on polarization of interfacial water, transmembrane potentials, and mechanisms for ion transport through bilayers are discussed. Our findings suggest that a range of published simulations results on the interaction of NaCl with phosphocholine bilayers have to be reconsidered and revised and that force field refinements are necessary for reliable simulation studies of membranes at physiological conditions on a molecular level.     

Interactions of Alkylphosphocholines with Model Membranes—The Langmuir Monolayer Study

Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔG ^exc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

Nacl flux in the flounder (Pseudopleuronectes americanus) intestine: Effects of ph and transport inhibitors

• 1.1. The effects of extracellular pH on Na⁺ and Cl⁻ absorption were studied in vitro in the small intestine of the winter flounder, Pseudopleuronectes americanus.
• 2.2. Reductions in bathing solution pH inhibited J_ms^na (mucosal-to-serosal flux) and J_net^na (net flux) (r = 0.90) and J_net^Cl (r = 0.92) [due to an increase in J_sm^Cl, (serosal-to-mucosal)] and decreased short circuit current (Isc).
• 3.3. Luminal bumetanide (0.1 mM) and amiloride (1 mM) inhibited Na⁺ and Cl⁻ absorption by reducing J_ms.
• 4.4. Luminal barium (5mM) and luminal copper (100 μM) decreased J_ms^Cl and increased J_sm^Cl.
• 5.5. We conclude that reductions in extracellular pH inhibit a luminal membrane NaCl absorptive process (Na⁺-K⁺-2Cl⁻) and stimulate an electrogenic Cl⁻ secretory process.

     

Potential difference responses to secretory K+, Cl− and Na+ changes in secreting and resting states of frog stomach

The effects of changes in secretory concentrations of K⁺, Cl⁻ and Na⁺ on transmembrane potential difference (PD) and resistance were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments histamine was present, and SCN and omeprazole gave similar results. Increase of K⁺ from 4 to 80 mM, decrease of Cl⁻ from 160 to 16 mM and decrease of Na⁺ from 156 to 15.6 mM gave, respectively, 10 min after the change, in the secreting fundus ΔPD = 7.6, 10.0 and −2.2 mV and in the resting fundus ΔPD = 4.3, 14.4 and 0 mV. With cimetidine and no histamine, increase of K⁺ from 4 to 80 mM gave a ΔPD which decreased to near zero after exposure to cimetidine for at least 30 min. For the same K⁺ change, replacement of cimetidine with SCN or omeprazole and without histamine maintained ΔPD near zero and subsequent addition of histamine with inhibitor present gave a ΔPD of about 12 mV. The change in ΔPD was attributed to histamine increasing the secretory membrane area, which results in an increase in K⁺ conductance. Increase in ΔPD in the resting fundus compared to the secreting fundus for a decrease from 160 to 16 mM Cl⁻ may be due to relatively little Cl⁻ entering the lumina from cells in the resting fundus, which would result in a greater change of the ratio intracellular Cl⁻/luminal Cl⁻ in the resting fundus than in the secreting fundus for the decrease in Cl⁻ studied.     

Disassembling and bleaching of chloride-free pharaonis halorhodopsin by octyl-beta-glucoside

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-d-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-d-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 mM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <--> OG-NpHR (disassembled) <--> intermediate --> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.     

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated ¹⁵N and ¹³CO chemical shifts of Val¹⁶ in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.     

K+ and Cl− Conductance of Arabidopsis thaliana Plasma Membrane at Depolarized Voltages

The ion currents, activated by depolarizations, across the plasma membrane of Arabidopsis thaliana cultured cells were studied by means of the patch-clamp technique in the whole-cell configuration. The electrical conductance of the membrane could be shifted from a cation to an anion conducting state by changes in the [K⁺]: [Cl⁻] ratio in the external medium. For ratios between 1:1 and 1:5 the currents were due to K⁺ efflux and for a ratio of 1:10 to Cl⁻ influx. In the cation conducting state the permeability ratio of K⁺ over NH⁺₄ and the alkali metal ions was: K⁺ ≅ NH>Na⁺ ≅ Li⁺ >Cs⁺. In the anion conducting state the permeability of NO⁻₃ was the same as that of Cl⁻. These channels were activated by depolarizations in the range of physiological potentials (-70/-80mV) and, either by mediating the efflux of cations or the influx of anions, they could function to re-hyperpolarize the membrane potential after depolarizations due to the influx of cations or of solutes cotransported with protons and/or to the inhibition of electrogenic pumps.     

Storage of abnormal oxidants ‘∑1’, ‘∑2’ and ‘∑3’ in photosynthetic water oxidases inhibited by Cl−-removal

The Cl⁻ requirement of photosynthetic O₂ evolution was studied by thermoluminescence measurements with purified Photosystem II-containing membrane particles from chloroplast thylakoids. When Cl anions had been removed from the particles either by an alkaline shock in a Cl⁻-free medium, or by treatment with SO²⁻₄, the pattern of the thermoluminescence emission after illumination with increasing numbers of flashes suggested that the oxidant storage in the water oxidase could only proceed up to the final step. The final step itself. i.e., the advance to the water-oxidizing S₄ state, apparently was blocked. An upward shift of the emission temperatures of the thermoluminescence bands was seen both in the absence and in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea, indicating that the stored oxidants had redox properties different from those of normal, Cl⁻-sufficient preparations. These properties were readily interconvertible by addition or removal of Cl⁻. We postulate that in Cl⁻-deficient water oxidases abnormal S₁, S₂ and S₃ states, symbolized as ∑₁, ∑₂ and ∑₃, respectively, are formed which are in a Cl⁻-dependent equilibrium with the corresponding normal S states. An oxidation of ∑₃ to a ∑₄ state is not possible. It is proposed that Cl⁻ controls the oxidation potential of the stored oxidants by regulating events associated with the binding and/or oxidative modification of water molecules at the water oxidase.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.