Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers

Charge-pulse relaxation experiments of valinomycin-mediated Rb⁺ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (k_R) and dissociation (k_D) of the ion-carrier complex as well as the rate constants of translocation of the complex (k_MS) and of the free carrier (k_S) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants k_S and k_MS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C₂₀ fatty acid from one to four resulted in an increase of k_S by a factor of seven and in an increase of k_MS by a factor of twenty-four. The stability constant K = k_R/k_D of the ion-carrier complex as well as the translocation rate constants k_S and k_MS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced k_R, k_MS and k_S up to seven-fold.     

The dimeric structure of the cytochrome bc1 complex prevents center P inhibition by reverse reactions at center N

Energy transduction in the cytochrome bc₁ complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c₁ reduction at varying quinol/quinone ratios in the isolated yeast bc₁ complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the b_H heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c₁ reduction indicated that the b_H hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of b_H heme reduction of 33%–55% depending on the quinol/quinone ratio. The extent of initial cytochrome c₁ reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the b_H hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the b_H hemes was explained using kinetics in terms of the dimeric structure of the bc₁ complex which allows electrons to equilibrate between monomers.     

Chloroplast Biogenesis 34: SPECTROFLUOROMETRIC CHARACTERIZATION IN SITU OF THE PROTOCHLOROPHYLL SPECIES IN ETIOLATED TISSUES OF HIGHER PLANTS

The fluorescence emission and excitation properties of protochlorophyll in etiolated cucumber (Cucumis sativus L.) cotyledons and primary bean (var. Red Kidney) leaves were characterized at 77 K. Contrary to previous studies, it appears that the short-wavelength protochlorophyll emission band consists of four fluorescent components, instead of only one nonphototransformable protochlorophyll. It was demonstrated that etiolated cucumber cotyledons synthesize and accumulate nontransformable protochlorophyll (E₄₄₀, F₆₃₀) as well as short-wavelength phototransformable protochlorophyll (E₄₃₃, F₆₃₃), (E₄₄₄, F₆₃₆), and (E₄₄₅, F₆₄₀). Long-wavelength phototransformable protochlorophyll (E₄₅₀, F₆₅₇) is also formed. In this context, E refers to the Soret excitation maxima and F refers to the red emission maxima of the protochlorophylls.     

Coiling of beta-pleated sheets

To form strongly twisted β-sheets, strands have to be coiled as well as twisted (Nishikawa &; Scherga, 1976). I show that strands coil in the appropriate right-handed direction if their main-chain torsion angles fulfil the following conditions: ψ_i ? ?φ_{i + 1}, ψ_{i + 1} > ?φ_{i + 2}, ψ_{i + 2} ? ?φ_{i + 3}, ψ_{i + 3} > ?φ_{i + 4}…Lactate dehydrogenase, pancreatic trypsin inhibitor, thermolysin and concanavalin A contain strongly twisted β-sheets and in each case the strands are coiled by their φ, ψ values fulfilling these conditions.     

The influence of external potassium on the inactivation of sodium currents in the giant axon of the squid, Loligo pealei

Isolated giant axons were voltage-clamped in seawater solutions having constant sodium concentrations of 230 mM and variable potassium concentrations of from zero to 210 mM. The inactivation of the initial transient membrane current normally carried by Na⁺ was studied by measuring the Hodgkin-Huxley h parameter as a function of time. It was found that h reaches a steady-state value within 30 msec in all solutions. The values of h _∞, τ_h, α_h,and β_h as functions of membrane potential were determined for various [K _o]. The steady-state values of the h parameter were found to be inversely related, while the time constant, τ_h, was directly related to external K⁺ concentration. While the absolute magnitude as well as the slopes of the h _∞ vs. membrane potential curves were altered by varying external K⁺, only the magnitude and not the shape of the corresponding τ_h curves was altered. Values of the two rate constants, α_h and β_h, were calculated from h_∞ and τ_h values. α_h is inversely related to [K_o] while β_h is directly related to [K_o] for hyperpolarizing membrane potentials and is independent of [K_o] for depolarizing membrane potentials. Hodgkin-Huxley equations relating α_h and β_h to E_m were rewritten so as to account for the observed effects of [K_o]. It is concluded that external potassium ions have an inactivating effect on the initial transient membrane conductance which cannot be explained solely on the basis of potassium membrane depolarization.     

Molecular typing of the self-incompatibility locus of Turkish sweet cherry genotypes reflects phylogenetic relationships among cherries and other Prunus species

Self-incompatibility of sweet cherry (Prunus avium L.) is controlled by the multiallelic S-locus. While many cultivars and wild accessions have been S-genotyped, only limited data are available on accessions native to the center of origin of this species. Therefore, this study was carried out to determine the S-genotype of 11 landrace cultivars and 17 local genotypes selected from populations growing wild at the Black Sea coast. Eleven sweet cherries (S ₁–S ₇, S ₁₀, and S ₁₂–S ₁₄) and some wild cherries (S ₁₇–S ₁₉, S _21/25, and S ₃₁) S-RNase alleles were detected. The results indicate that Turkish cultivars represent a broader gene pool as compared with international cultivars. A new (S ₃₇) and a doubtful allele (provisionally labelled as S _7m) as well as the sour cherry S ₃₄-allele were identified in sweet cherry. These data and others (SSR variants within the S ₁₃-RNase introns) confirmed that allele pools of sweet and sour cherries in the Black Sea region are overlapping. A new cross-incompatibility group, XLV (S ₂ S ₁₈), was also proposed. Allele-specific primers were designed for S ₁₇–S ₁₉, S _21/25, S ₃₄, and S ₃₇. A phylogenetic analysis of the cherry S ₃₁-RNase and its trans-specific sister alleles reliably mirrored the assumed length of the time period after the divergence of species in the subgenera Cerasus and Prunophora. Most variations (insertions/deletions and single-nucleotide polymorphisms) in the S-RNase gene were silent and, hence, have not been exposed to natural selection. The results are discussed from the aspects of S-allele evolution and phylogenetic relationships among cherries and other Prunus species.     

Modelling ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants: I. regulation of pre-industrial atmosphere

In closed systems, the O₂ compensation point (Γ_O) was previously defined as the upper limit of O₂ level, at a given CO₂ level, above which plants cannot have positive carbon balance and survive. Studies with ¹⁸O₂ measure the actual O₂ uptake by photorespiration due to the dual function of Rubisco, the enzyme that fixes CO₂ and takes O₂ as an alternative substrate. One-step modelling of CO₂ and O₂ uptakes allows calculating a plant specificity factor (Sp) as the sum of the biochemical specificity of Rubisco and a biophysical specificity, function of the resistance to CO₂ transfer from the atmosphere to Rubisco. The crossing points (Cx, Ox) are defined as CO₂ and O₂ concentrations for which O₂ and CO₂ uptakes are equal. It is observed that: (1) under the preindustrial atmosphere, photorespiration of C3 plants uses as much photochemical energy as photosynthesis, i.e. the Cx and Ox are equal or near the CO₂ and O₂ concentrations of that epoch; (2) contrarily to Γ_C, a Γ_O does not practically limit the plant growth, i.e. the plant net CO₂ balance is positive up to very high O₂ levels; (3) however, in a closed biosystem, Γ_O exists; it is not the limit of plant growth, but the equilibrium point between photosynthesis and the opposite respiratory processes; (4) a reciprocal relationship exists between Γ_O and Γ_C, as unique functions of the respective CO₂ and O₂ concentrations and of Sp, this invalidates some results showing two different functions for Γ_O and Γ_C, and, consequently, the associated analyses related to greenhouse effects in the past; (5) the pre-industrial atmosphere levels of O₂ and CO₂ are the Γ_O and Γ_C of the global bio-system. They are equal to or near the values of Cx and Ox of C3 plants, the majority of land plants in preindustrial period. We assume that the crossing points represent favourable feedback conditions for the biosphere-atmosphere equilibrium and could result from co-evolution of plants-atmosphere-climate. We suggest that the evolution of Rubisco and associated pathways is directed by an optimisation between photosynthesis and photorespiration.     

Cytochrome c6B of Synechococcus sp. WH 8102 – Crystal structure and basic properties of novel c6-like family representative

Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c₆ participates in electron transfer from cytochrome b₆f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c_6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c₅₅₀ is well known element of photosystem II. However, function of cytochromes marked as c_6B, c_6C and c_M as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c_6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c₆, are slightly red-shifted α band of UV–Vis spectrum as well as relatively low midpoint potential (113.2 ± 2.2 mV). Although, physiological function of cytochrome c_6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b₆f complex and photosystem I.     

Homogeneous CO hydrogenation and transfer hydrogenation catalyzed by ruthenium(II) complexes containing optically active Ph2PCH(Ph)CH(Me)NH2 and 1,2-C5H8(PPh2)2 ligands

Combination of (1S,2S)-cyclopentanediylbis(diphenylphosphine) with [Ru(η⁴-C₈H₁₂){η³-(CH₂)₂CMe}₂] afforded the chelate complex [Ru{η³-(CH₂)₂CMe}₂{(1S,2S)-C₅H₈(PPh₂)₂}] (1), which gave (OC-6-13)-[RuCl₂{(1S,2S)-C₅H₈(PPh₂)₂}{(1S,2S)-Ph₂PCH(Ph)CH(Me)NH₂}] (2) upon reaction with methanolic HCl in acetone, followed by the addition of the β-aminophosphine in DMF. The (P ∩ N)₂-chelated complexes (OC-6-13)-[RuCl₂{(1S,2S)-Ph₂PCH(Ph)CH(Me)NH₂}₂] (3) and (OC-6-13)-[RuCl₂{(1R,2S)-Ph₂PCH(Ph)CH(Me)NH₂}₂] (4) resulted from RuCl₃ · 3H₂O and the P,N ligands under reducing conditions. The crystal structures of 3 and 4 were determined by single-crystal X-ray diffraction. Following activation by KOBu-t in isopropanol, compounds 2–4 catalyzed the enantioselective transfer hydrogenation of acetophenone with i-PrOH as the hydrogen source as well as the direct hydrogenation of the ketone by H₂ in low to moderate e.e. (up to 67%).     

Synthesis, structure, spectroscopic and magnetic characterization of a novel spin-crossover iron(II) complex with 1-cyclopropyltetrazole ligands

For the first time a 1-cyclopropyl substituted tetrazole (C₃tz) has been used as a potential ligand for iron(II) spin-transition complexes. The complexation of 1-cyclopropyltetrazol with iron(II) tetrafluoroborate yielded a fine powdered product of [Fe(C₃tz)₆](BF₄)₂ being poorly soluble in most common solvents. Single crystals of complex were grown in situ from a solution of ligand and iron(II) hexafluorophosphate, which yielded a hexagonal prismatic crystalline product of [Fe(C₃tz)₆](PF₆)₂. A comparison of XRPD data of the homologues [Fe(C₃tz)₆](BF₄)₂ and [Fe(C₃tz)₆](PF₆)₂ proves them to be homeotypic. The thermally induced spin-crossover phenomenon of [Fe(C₃tz)₆](BF₄)₂ complex shows very abrupt spin transitions, with a spin-crossover temperature T_1/2 ≈ 180 K which is found to be ≈50 K above the T_1/2 of all known iron(II) complexes with n-alkyltetrazoles as ligands. The T_1/2 was determined by temperature-dependent ⁵⁷Fe-Mössbauer, far FT-IR and UV-Vis-NIR spectroscopy as well as temperature dependent magnetic susceptibility measurements (SQUID).     

The development and initial validation of a virtual dripping sweat rate and a clothing wetness ratio for use in predictive heat strain models

This paper applies the heat balance equation (HBE) for clothed subjects as a linear function of mean skin temperature (t _sk ) by a new sweating efficiency (η _sw ) and an approximation for the thermoregulatory sweat rate. The equation predicting t _sk in steady state conditions was derived as the solution of the HBE and used for a predictive heat strain scale. The heat loss from the wet clothing (WCL) area was identified with a new variable of ‘virtual dripping sweat rate VDSR’ (S _wdr ). This is a subject’s un-evaporated sweat rate in dry clothing from the regional sweat rate exceeding the maximum evaporative capacity, and adds the moisture to the clothing, reducing the intrinsic clothing insulation. The S _wdr allowed a mass balance analysis of the wet clothing area identified as clothing wetness (w _cl ). The w _cl was derived by combining the HBE at the WCL surface from which the evaporation rate and skin heat loss from WCL region are given. Experimental results on eight young male subjects wearing typical summer clothing, T-shirt and trousers verified the model for predicting t _sk with WCL thermal resistance (R _cl,w ) identified as 25 % of dry clothing (R _cl,d ).     

Differential stability of trp messenger RNA synthesized originating at the trp promoter and pL promoter of lambda trp phage.

The trp operon translocated into the early region of phage λ can be transcribed under the control of two promoters, the authentic trp promoter (p_Ttrp mRNA) and the p_L promoter of the N gene (p_Ltrp mRNA) (Imamoto &; Tani, 1972; Segawa &; Imamoto, 1974). The p_Ltrp mRNA has a 5′-terminal λ N message. The functional and chemical stability of trp segments in these mRNA species have been assayed. To determine trp mRNA from λtrp, appropriate φ80trp DNAs were used as a DNA complement in DNA-RNA hybridization assays.When formation of mRNA is inhibited, the capacity to serve as template for enzyme synthesis decays at a comparable rate for p_L and p_Ttrp mRNA, and p_Ltrp mRNA seems to be translated as efficiently as is the normal p_Ttrp mRNA. In contrast to this similar functional stability, p_Ltrp mRNA shows a more than tenfold greater chemical stability than p_Ttrp mRNA. (p_Ttrp mRNA is degraded at the same rate as trp mRNA in uninfected bacteria. Bulk host mRNA also decays at its normal rate in cells infected with λtrp.)On the basis of those and more extensive experiments including the sedimentation analysis of those stabilized trp mRNA molecules, it is inferred that (1) the rate-limiting step to initiate bulk mRNA degradation is determined by a sequence located at or near the 5′ end of the messenger RNA; and (2) functional inactivation of each messenger is regulated independently of bulk chemical degradation of the message.Stabilization of the trp mRNA produced from the p_L promoter increases with time after phage infection. Thus, the stabilization requires a modification of the decay trigger, possibly by a phage-specific protein such as a nuclease or the N and/or tof gene that might bind to the mRNA.     

Hydrogen sulfide is a mediator in H2O2-induced seed germination in Jatropha Curcas

Hydrogen peroxide (H₂O₂), a second messenger, plays a vital role in seed germination and plant growth, development as well as the acquisition of stress tolerance, while hydrogen sulfide (H₂S) is considered as a new emerging cell signal molecule in higher plants. In the present study, soaking of H₂O₂ greatly improved germination percentage of Jatropha curcas seeds, stimulated the increase of l-cysteine desulfhydrase activity, which in turn induced accumulation of H₂S. On the contrary, pretreatment of aminooxyacetic acid (AOA), inhibitor of H₂S biosynthesis, eliminated H₂O₂ stimulated the increase of activity of l-cysteine desulfhydrase and accumulation of H₂S as well as improvement of germination percentage. In addition, exogenously applied H₂S also could improve germination percentage of seeds of J. curcas. These results suggested that pretreatment of H₂O₂ could improve germination percentage of J. curcas seeds and this improvement was mediated by H₂S.     

Concentration Dependence of Permeability Coefficients to an Electrolyte Component across Bovine Lens Capsule In Vitro

Theoretical and experimental studies have been made on permeability coefficients to various kinds of electrolyte across lens capsules that are dissected from bovine eyes and that are found to be positively fixed charged membranes from our experiments of membrane potentials. The differential permeability coefficient, P_m, is defined as J_s = P_m(C₂ - C₁), where J_s is the flux of an electrolyte component in moles per sec across unit area of the lens capsule that separates two aqueous solutions of the same electrolyte at different concentrations, C₂ and C₁. Various types of strong electrolytes were studied; KCl, NaCl, Cacl₂, MgSO₄, MgCl₂ and LaCl₃. It was found that at C₂/C₁ = constant, P_m decreases to zero as C₂ decreases and P_m increases to a limiting value, (P_m)^∞, that is characteristic for the system of the salt used and the membrane as C₂ increases, despite of electrolytes. We assumed in theory that single ion activity coefficients of co-ion and gegen-ion are ideal, that the systems studied are in electric neutrality, that the fixed charge density of the membrane is independent of concentrations C₂, and that Donnan equilibrium holds between the bulk solution and membrane surface. Although the concentration-dependent changes of P_m were quantitatively different depending on the type of electrolyte used, general agreement between theory and experiment was obtained over a wide range of concentrations except for the case of very dilute solutions.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

Production of hemicellulases by three fungi pathogenic to sugar cane

Three fungal pathogens, Ceratocystis paradoxa (CP), Cephalosporium sacchari (CS), and Marasmius sacchari (MS) were screened for the production of hemicellulose-degrading enzymes (hemicellulases) by induction on bagasse hemicellulose B, and on a commercial preparation of hemicellulose (crude xylan). All three pathogen initially grew poorly on hemicellulose B and “crude xylan” as carbon source. Profuse growth was induced, however, by using mixtures of hemicellulose B and sucrose in the culture media for CP and CS until the organisms were capable of growing on media containing only hemicellulose B. These isolates were classified as CS₁ and CP₁. Profuse growth occurred when CS and CP were grown on carboxymethylcellulose (CMC) and also when these cultures were transferred to media containing only hemicellulose B. These isolates were classified as CS₂ and CP₂. When the above four isolates were grown on hemicellulose B as carbon source in submerged liquid culture, only CP₁ did not produce any extra-cellular hemicellulase(s), and CP₂ produced the highest yield of enzyme. CS₂ and CP₂ also produced extra-cellular CM-cellulase(s). The CP₂-culture isolate was selected for the study of conditions for the optimal production of extra-cellular hemicellulase(s). A preliminary study of the action of enzymes from CS and CP isolated on hemicellulose is reported.     

Relation between mechanical foam-breaking difficulty and the foaming characteristics of solutions

Using a small foaming apparatus, liquid foaming characteristics such as liquid holdup in foamate flow (φ_t), rate of liquid drainage from foam (ν), foam velocity (u_f) and foam size (d_f) were examined for various liquids. A parameter φ_f, incorporating φ_t, ν and u_f, was obtained. The relationship among φ_f, d_f and the liquid holdup in foam, φ_b, in the actual gas-bubbling systems under foam control was expressed in the form of φ_b ∞ φ_fd_f^1.83, regardless of the type of foaming liquid or its concentration. This relationship was useful for the prediction of φ_b as related to mechanical foam-breaking difficulty.     

Characterization and properties of an inulinase from a thermophilic bacteria

The inulinase of the thermophilic bacterial strain LCB41 (Bacillus sp.) was produced in fermentor using a mineral medium containing inulin as carbon source. The enzyme content was as high as the known inulinase producers and most of the activity was found in the culture medium. The enzyme was stable at high temperature and active at neutral and slightly basic pH. Fructose is liberated as the sole reaction product of inulin hydrolysis, classifying the enzyme as an exoinulinase. Inulin and sucrose were both hydrolyzed at appreciable rates with an (I/S) ratio of 0·40 and (V_m/K_m)₁/(V_m/K_m)_S = 9·9. The enzyme was less inhibited than yeast invertase or Kluyveromyces fragilis inulinase at high sucrose concentrations. The inulinase of strain LCB41 is a good candidate for industrial hydrolysis of inulin or sucrose.     

Homogeneous oxidative coupling catalysts. Mechanism of conversion of 2,6-dimethylphenol [DMP] to 3,3′,5,5′-tetramethyl-4,4′-diphenoquinone [DPQ] by [(Pip)nCuX]4O2 (n = 1 or 2, Pip = piperidine, X = Cl, Br or I) in aprotic media

This paper represents the mechanism of the second half of the catalytic cycle, Scheme 1, which represents the conversion of 2,6-dimethylphenol [DMP] to 3,3′,5,5′-tetramethyl, 4,4′-diphenoquinone [DPQ] by homogenous oxidative coupling catalysts [(Pip)_nCuX]₄O₂ in aprotic media. The mechanism can be represented as a pre-equilibrium, K, between the catalyst and 2,6-dimethylphenol to form a complex intermediate which is converted into the activated complex through the rate determining step, k₂, to form the final products. The observed pseudo first-order rate constant is given by k_obs = K k₂[DMP]^y/(1 + K[DMP]^y). When the coordination number around copper(II) is equal to five as in [(Pip)CuX]₄O₂, the system suffers from kinetic saturation due to strong complex formation between catalyst and [DMP] and therefore K[DMP]^y > 10 and k_obs = k₂. Kinetic saturation has been avoided by using six coordinate copper(II) as in [(Pip)₂CuX]₄O₂. The influence of the coordination saturation of copper(II) in [(Pip)₂CuX]₄O₂ helps to evaluate both thermodynamic and kinetic parameters for the system as well as for the structure of the activated complex, (y = 2), which consists of one [(Pip)₂CuX]₄O₂ and two [DMP]. Reduction of copper(II) to copper(I) has been suggested as a rate determining step due to halogen, X, and solvent effects.