Inner voltage clamping. A method for studying interactions among hydrophobic ions in a lipid bilayer

Ketterer, et al. (1971) have suggested that a combination of electrostatic and chemical interactions may cause hydrophobic ions absorbed within a bilayer lipid membrane to reside in two potential wells, each close to a membrane surface. The resulting two planes of charges would define three regions of membrane dielectric: two identical outer regions each between a plane of absorbed charges and the plane of closest approach of ions in the aqueous phase; and the inner region between the two planes of adsorbed charges. The theory describing charge translocation across the inner region is based on a simple three-capacitor model. A significant theoretical conclusion is that the difference between the voltage across the inner region, V_i, and the voltage across the entire membrane, V_m, is directly proportional to the amount of charge that has flowed in a voltage clamp experiment. We demonstrate that we can construct an “inner voltage clamp” that can maintain, with positive feedback, a constant inner voltage, V_i. The manifestation of proper feedback is that the clamp current (after a voltage step) will exhibit pure (i.e., single time-constant) exponential decay, because the voltage dependent rate constants governing translocation will be independent of time. The “pureness” of the exponential is maximized when the standard deviation of the least-square fit of the appropriate exponential equation to the experimental data is minimized. The concomitant feedback is directly related to the capacitances of the inner and outer membrane regions, C_i and C_o.

Experimental results with tetraphenylborate ion adsorbed in bacterial phosphatidylethanolamine/n-decane bilayers indicate C_i ~ 5 · 10^-7F/cm² and C_o ≈ 5 · 10^-5F/cm².

     

Intensity of nitrification in soil as a function of time and soil moisture

Proceeding from three previously derived expressions for the intensity of nitrification in soil as a function of time (logΣN=K.logt+q), as a function of incubation moisture (logΣN=A.pF _i+B), as a function of initial moisture (logΣN=C.pF _v+D), it was shown that the nitrification intensity as a function of time and of moisture can be expressed by the bilinear function log ΣN=a.pF _i.logT+b.pF _i+c.logt+d; as a function of time and of initial moisture by the bilinear function logΣ=N=a.pF _v.logt+b.pF _v+c.logt+d; as a function of initial and incubation moisture by the bilinear function log ΣN=a.pF _ipF _v+b.pF _i+c.pF _v+d. The intensity of nitrification as a function of time, incubation moisture and initial moisture may be expressed by the multilinear function log ΣN=a.pF _i.pF _v.logt+b.pF _i.pF _v+c.pF _i.logt+d.pF _v.logt+e .pF _i+f.pF _v=g.logt+h. This function is valid for all the incubation moistures lying between pF _i 3.0 and 4.0 and for all initial moistures between 3.5 and 5.9 provided that the incubation temperature remains constant.     

A Simple Algorithm for Finding All k-Edge-Connected Components

The problem of finding k-edge-connected components is a fundamental problem in computer science. Given a graph G = (V, E), the problem is to partition the vertex set V into {V ₁, V ₂,…, V _h}, where each V _i is maximized, such that for any two vertices x and y in V _i, there are k edge-disjoint paths connecting them. In this paper, we present an algorithm to solve this problem for all k. The algorithm preprocesses the input graph to construct an Auxiliary Graph to store information concerning edge-connectivity among every vertex pair in O(Fn) time, where F is the time complexity to find the maximum flow between two vertices in graph G and n = ∣V∣. For any value of k, the k-edge-connected components can then be determined by traversing the auxiliary graph in O(n) time. The input graph can be a directed or undirected, simple graph or multigraph. Previous works on this problem mainly focus on fixed value of k.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Editorial

The voltage dependence of the rat renal type II Na⁺/P_i cotransporter (NaP_i-2) was investigated by expressing NaP_i-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (P_i) induced inward currents (I_p) in the presence of 96 mM Na⁺ over the potential range −140 ≤ V ≤ +40 mV. With P_i as the variable substrate, the apparent affinity constant (K _m ^Pi) was strongly dependent on Na⁺, increasing sixfold for a twofold reduction in external Na⁺. K _m ^Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na⁺. The Hill coefficient was close to unity and the predicted maximum I_p (I_pmax) was 40% smaller at 50 mM Na⁺. With Na⁺ as the variable substrate, K _m ^Na was weakly dependent on both P_i and voltage, the Hill coefficient was close to 3 and I_pmax was independent of P_i at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na⁺-dependent manner, indicating the existence of uncoupled Na⁺ slippage. Voltage steps induced pre–steady state relaxations typical for Na⁺-coupled cotransporters. NaP_i-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na⁺, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V_0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e⁻. V_0.5 became more negative as Na⁺ was reduced. P_i suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V_0.5 to depolarizing potentials and suppressed relaxations in the absence of Na⁺, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na⁺ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na⁺ binding step.     

Segmental flexibility of ribosomal protein S1 bound to ribosomes and Q beta-replicase

The protonization pattern of the endogenous donor component D₁ which feeds electrons directly into chl-a⁺_II has been analyzed in Tris-washed inside-out thylakoids with the aid of appropriate pH-indicators. It was found that under repetitive flash excitation the amount of protons released is proportional to the extent of D₁-oxidation, depending on the time between the flashes. The kinetics of D₁-oxidation (being practically the same as in normal Tris-washed chloroplasts) are faster than the proton release by two orders of magnitude. The results lead to the conclusion that D₁ is protonized in the reduced state with pK(D^ox₁) < 5 and becomes deprotonized in the oxidized state with pK(D^red₁) ? 8. The proton release is kinetically limited by a transport barrier. Implications on the interpretation of the proton release pattern in preparation with intact water oxidation are discussed.     

Physical Parameters of a Reactor-Stellarator with Small Ripples of the Helical Magnetic Field

The paper describes the calculation data on the physical parameters of a reactor-stellarator, where the nonuniformities of the helical field are smaller than the toroidal magnetic field nonuniformities: ε_h < ε_t. Unlike the previous studies, where the ion-component transport coefficients had the collision frequency dependence proportional to ν^1/2, this being equivalent to the ε_h > ε_t case, in the present calculations, these coefficients were assumed to be in proportion to the first power of the collision frequency, D_i ∝ ν for ν_eff < 2ω_E, and to D_i ∝ ν^?1 for the inverse inequality. Here, ω_E is the rotation frequency of plasma in the radial electric field. As before, the plasma electrons corresponded to the mode of D_e ∝ ν^?1. As initial parameters for numerical calculations, a reactor with R = 8 m, r_p = 2 m, and B₀ = 5 Т was taken. A numerical code was used to solve the set of equations that describes the plasma space?time behavior in the reactor-stellarator under the conditions of equal diffusion fluxes. The start of reactor operation in the mode of thermonuclear burning was provided by heating sources with a power of several tens of megawatts. Steady-state operating conditions of a self-sustained thermonuclear reaction were attained by maintaining the plasma density through DT fuel pellet injection into the plasma.     

Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O.

Between 20 and 40 °C D₂O inhibits the hydrolytic activity of soluble mitochondrial ATPase F₁. The effect of D₂O is proportional to its concentration in the incubation mixture and at nearly 100% D₂O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D₂O is mainly on the V of the reaction. At temperatures above 45 °C, D₂O does not inhibit the activity. D₂O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F₁. The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F₁ increases as the enzyme becomes inactivated by low temperatures; in D₂O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F₁ and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D₂O may be employed with success in the isolation and purification of labile enzymes.     

The area under the function: an index for selecting desirable genotypes

The linear regression approach has been widely used for selecting high-yielding and stable genotypes targeted to several environments. The genotype mean yield and the regression coefficient of a genotype's performance on an index of environmental productivity are the two main stability parameters. Using both can often complicate the breeder's decision when comparing high-yielding, less-stable genotypes with low-yielding, stable genotypes. This study proposes to combine the mean yield and regression coefficient into a unified desirability index (D _i). Thus, D _i is defined as the area under the linear regression function divided by the difference between the two extreme environmental indexes. D _i is equal to the mean of the i ^th genotype across all environments plus its slope multiplied by the mean of the environmental indexes of the two extreme environments (symmetry). Desirable genotypes are those with a large D _i. For symmetric trials the desirability index depends largely on the mean yield of the genotype and for asymmetric trials the slope has an important influence on the desirability index. The use of D _i was illustrated by a 20-environments maize yield trial and a 25-environments wheat yield trial. Three maize genotypes out of nine showed values of D _i 's that were significantly larger than a hypothetical, stable genotype. These were considered desirable, even though two of them had slopes significantly greater than 1.0. The results obtained from ranking wheat genotypes on mean yield differ from a ranking based on D _i .     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Convenient experimental determination of adsorption isotherms in a Hydrophobic Interaction Chromatography system

Summary HPLC was combined with a packable microbore guard column to obtain the adsorption isotherm of lysozyme in a Hydrophobic Interaction Chromatography system. The equipment configuration enabled isotherm determination of the protein on a relatively low pressure chromatographic media (TosoHaas 650M Phenyl).Notation C_m,i is the mobile phase concentration of protein. (M/L³ _(liquid)) - C_m,0 =0 - C_s,i is the stationary phase concentration of protein. It is the concentration of protein on the chromatographic media. (M/L³ _(solid)) - C_s,0 =0 - M,L is the dimensions mass and length - V_r,i is the retention volume of the peak front that corresponds to a mobile phase protein on the concentration C_m,i. (L³ _(liquid)) - i i is a counter that is used to keep track of C_m, C_s, and V_r.For example, i=1 in the term C_m,i denotes the first, and lowest, mobile phase protein concentrations are described by higher values of i. - V_d is the system dead volume. It consists of all of the system volume that the mobile phase "sees" or contacts, includingchromatographic media interparticle and pore volume. (L³ _liquid) - V_s the stationary phase volume. V_s is the nonporous bead volume. For porous beads, V_s is the bead volume - the porevolume. (L³ _(solid)) - V_e is the empty column volume. (L³ _liquid) - V_m is the packed column mobile phase volume and consists of the pore volume and the excluded volume. (L³ _(liquid)) - V_{e system} is the empty column system volume. (L³ _(liquid)) - V_frit the volume of mobile phase that fills the column frits. (L³ _(liquid)) - V_woc the system volume without the column connected. (L³ _(liquid))     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pK_a values of 6.1 and 8.6. A dissociable group with a pK_a of 8.5 on the enzyme-substrate complex affects the pK_m for isocitrate. The pK_i for homoisocitrate is affected in a like manner. The pH dependence of the pK_i's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pK_m of isocitrate below pH 7.3, but is markedly different above this pH. Both the K_m for isocitrate and the K_i for succinate were dependent upon Mg²⁺ concentration. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pK_a of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pK_i for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pK_m for isocitrate, on the enzyme-glyoxylate-inhibitor complex.     

Insights into the nature of Mo(V) species in solution: Modeling catalytic cycles for molybdenum enzymes

The tris(pyrazolyl)borate and related tripodal N-donor ligands originally developed by Trofimenko stabilize mononuclear compounds containing Mo^VIO₂, Mo^VIO, Mo^VO, and Mo^IVO units and effectively inhibit their polynucleation in organic solvents. Dioxo-Mo(VI) complexes of the type LMoO₂(SPh), where L = hydrotris(3,5-dimethylpyrazol-1-yl)borate (Tp^∗), hydrotris(3-isopropylpyrazol-1-yl)borate (Tp^iPr), and hydrotris(3,5-dimethyl-1,2,4-triazol-1-yl)borate (Tz) and related derivatives are the only model systems that mimic the complete reaction sequence of sulfite oxidase, in which oxygen from water is ultimately incorporated into product. The quasi-reversible, one-electron reduction of Tp^∗MoO₂(SPh) in acetonitrile exhibits a positive potential shift upon addition of a hydroxylic proton donor, and the magnitude of the shift correlates with the acidity of the proton donor. These reductions produce two Mo(V) species, [Tp^∗Mo^VO₂(SPh)]⁻ and Tp^∗Mo^VO(OH)(SPh), that are related by protonation. Measurement of the relative amounts of these two Mo(V) species by EPR spectroscopy enabled the pK_a of the Mo^V(OH) unit in acetonitrile to be determined and showed it to be several pK_a units smaller than that for water in acetonitrile. Similar electrochemical-EPR experiments for Tp^iPrMoO₂(SPh) indicated that the pK_a for its Mo^V(OH) unit was ∼1.7 units smaller than that for Tp^∗Mo^VO(OH)(SPh). Density functional theory calculations also predict a smaller pK_a for Tp^iPrMo^VO(OH)(SPh) compared to Tp^∗Mo^VO(OH)(SPh). Analysis of these results indicates that coupled electron-proton transfer (CEPT) is thermodynamically favored over the indirect process of metal reduction followed by protonation. The crystal structure of Tp^iPrMoO₂(SPh) is also presented.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Scorpion venom complexity fractal analysis. Its relevance for comparing venoms

We analyzed the venom elution pattern of 15 scorpions species. Data were scanned at 1 Hz and stored digitally. Approximate fractal dimension (D) [Sevcik (1998)] was calculated for minutes 0-60 of the elutions. D was calculated for either the whole time range, or calculated using a window of 500 points, which was displaced by one time increment recursively, and stored [(t_i,D_i) sets]. We avoid the term complexity as much as possible since defining complexity is difficult; instead we propose the term contortedness and represent it by the variable Q=D−1. To compare venom contortednesses of different species, a phase plot with their (t_i,Q_i) sets was constructed and determination coefficient (d_s) were calculated squaring the Spearman rank correlation coefficient. (t_i,Q_i) sets of several elutions of the same specie were averaged and compared with other species finding that some were amazingly similar (Tityus clathratus vs Tityus caripitensis, d_s = 0.813). Tityus discrepans was similar to 6 of 8 species of the same genus (d_s ranging from 0.23 to 0.49), and also similar to Centruroides gracilis and Chactas laevipes (d_s 0.54 and 0.49, respectively). Serendipitously,T. discrepans was chosen many years ago to produce anti-Tityus antivenom in Venezuela; perhaps the clinical success in neutralizing the venom of the other known Venezuelan Tityus, stems from the mimetism of this venom with the remaining species’ venom.