Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ϵ and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG₀) and the affinity change for ACh that increases the open channel probability (ΔG_B). The effects of mutations of ProD2 (ϵPro-121/δPro-123) were greater than those of its neighbor (ϵPro-120/δPro-122) and were greater at α-ϵ versus α-δ. The main consequence of the congenital myasthenic syndrome mutation ϵProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔG_B less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG₀). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔG_B^ACh less favorable) by ≥2 kcal/mol at α-ϵ and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ϵ and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at ϵProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ϵ or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately −5 kcal/mol).     

Electrogenic ATPase Activity on the Peribacteroid Membrane of Soybean (Glycine max L.) Root Nodules

Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (ΔΨ), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive ΔΨ across the peribacteroid membrane was dependent upon ATP, inhibited by N,N′-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO₃⁻ > NO₂⁻ > Cl⁻ > acetate⁻ > malate⁻. The observation that 10 millimolar NO₃⁻ completely dissipated the membrane potential was of particular interest in view of the fact that NO₃⁻ inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.     

Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

Immobilised Histidine Tagged β 2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β ₂-adrenoceptor (β ₂-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β ₂-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β ₂-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10³/M for ephedrine and (3.80±0.02) ×10³/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10⁻⁴ M. Thermodynamic studies showed that the binding of the two compounds to β ₂-AR was a spontaneous reaction with exothermal processes. The ΔG^θ, ΔH^θ and ΔS^θ for the interaction between ephedrine and β ₂-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β ₂-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.     

Probing the Interaction of a Therapeutic Flavonoid,Pinostrobin with Human Serum Albumin: Multiple Spectroscopic and Molecular Modeling Investigations

Interaction of a pharmacologically important flavonoid, pinostrobin (PS) with the major transport protein of human blood circulation, human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity (1.03 × 10⁵ M⁻¹ at 25°C) between PS and HSA with a 1∶1 stoichiometry. Thermodynamic analysis of the binding data (ΔS = +44.06 J mol⁻¹ K⁻¹ and ΔH = −15.48 kJ mol⁻¹) and molecular simulation results suggested the involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. Both secondary and tertiary structural perturbations in HSA were observed upon PS binding, as revealed by intrinsic, synchronous, and three-dimensional fluorescence results. Far-UV circular dichroism data revealed increased thermal stability of the protein upon complexation with PS. Competitive drug displacement results suggested the binding site of PS on HSA as Sudlow’s site I, located at subdomain IIA, and was well supported by the molecular modelling data.     

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.     

Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Δ290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Δ290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 × 10⁻⁸ M versus 3.2 × 10⁻⁶ M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Δ290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster k_on rather than to a slower k_off. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 × 10⁻⁵ M) than did gD1(306t) due to a more rapid k_off. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Examination of the Kinetics of Herpes Simplex Virus Glycoprotein D Binding to the Herpesvirus Entry Mediator, Using Surface Plasmon Resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (K_D) of 3.2 × 10⁻⁶ M and gD2(306t) had a K_D of 1.5 × 10⁻⁶ M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Δ290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (K_D = 3.3 × 10⁻⁸ M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Δ290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline,Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)₄, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K_i values of 2.01 × 10⁻⁵ and 1.84 × 10⁻⁶ m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (−TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K_d value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

The Ileal Lipid Binding Protein Is Required for Efficient Absorption and Transport of Bile Acids in the Distal Portion of the Murine Small Intestine

The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6^−/− mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05) in female but not male Fabp6^−/− mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05) but not in male Fabp6^−/− mice. The amount of [³H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (P<0.025) higher for female Fabp6^−/− mice whereas it was 57.3% (P<0.01) lower for male Fabp6^−/− mice, compared to wild-type mice. The retained fraction of the [³H]TCA localized in the small and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6^−/− mice relative wild-type mice, whereas no changes were seen in female Fabp6^−/− mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6^−/− mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.     

Preference of carbon absorption determines the competitive ability of algae along atmospheric CO2 concentration

Although many studies have focused on the effects of elevated atmospheric CO₂ on algal growth, few of them have demonstrated how CO₂ interacts with carbon absorption capacity to determine the algal competition at the population level. We conducted a pairwise competition experiment of Phormidium sp., Scenedesmus quadricauda, Chlorella vulgaris and Synedra ulna. The results showed that when the CO₂ concentration increased from 400 to 760 ppm, the competitiveness of S. quadricauda increased, the competitiveness of Phormidium sp. and C. vulgaris decreased, and the competitiveness of S. ulna was always the lowest. We constructed a model to explore whether interspecific differences in affinity and flux rate for CO₂ and HCO₃ ⁻ could explain changes in competitiveness between algae species along the gradient of atmospheric CO₂ concentration. Affinity and flux rates are the capture capacity and transport capacity of substrate respectively, and are inversely proportional to each other. The simulation results showed that, when the atmospheric CO₂ concentration was low, species with high affinity for both CO₂ and HCO₃ ⁻ (HCHH) had the highest competitiveness, followed by the species with high affinity for CO₂ and low affinity for HCO₃ ⁻ (HCLH), the species with low affinity for CO₂ and high affinity for HCO₃ ⁻ (LCHH) and the species with low affinity for both CO₂ and HCO₃ ⁻ (LCLH); when the CO₂ concentration was high, the species were ranked according to the competitive ability: LCHH > LCLH > HCHH > HCLH. Thus, low resource concentration is beneficial to the growth and reproduction of algae with high affinity. With the increase in atmospheric CO₂ concentration, the competitive advantage changed from HCHH species to LCHH species. These results indicate the important species types contributing to water bloom under the background of increasing global atmospheric CO₂, highlighting the importance of carbon absorption characteristics in understanding, predicting and regulating population dynamics and community composition of algae.