Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Novel findings on the copper catalysed oxidation of cysteine

Summary The oxidation of cysteine (RSH) has been studied by using O₂, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200M Cu^II to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-Cu^I-SR) is responsible for this absorbance. In the absence of O₂ the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu^II enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-Cu^I-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.Abbreviations RSH cysteine - RS^– cysteine in the thiolate form - RS^· thiyl radical of cysteine - RSSR cystine - Cyt c cytochrome c - SOD superoxide dismutase - NBT nitro blue tetrazolium - NBF nitro blue formazan - DTNB 5,5-dithiobis-2-nitrobenzoic acid - DTPA diethylenetriaminepentaacetic acid Dedicated to prof. A. Ballio ob the occasion of his 75th birthday.     

Synthesis of some racemicγ-fluoro-α-amino acids

Summary Versatile three-step procedures for syntheses of seven racemi-fluoro-a-amino acids are described. Alkylation oftert-butyl N-(diphenylmethylene) glycinate with 1-bromo-2-fluoroalkanes gave N-protected aminoacid esters both in anhydrous medium using lithium-diisopropylamide as base at low temperature or in a two phase system of 50% aqueous sodium hydroxide and methylene chloride with triethylbenzylammonium chloride as the phase transfer catalyst at room temperature. Subsequent two-step deprotection with citric acid and hydrochloric acid gave the title compounds in 13–33% overall yields.Dedicated to Professor Dr.mult., Dr.h.c. Alois Haas on the occasion of his 65th birthday     

On the RNA World: Evidence in Favor of an Early Ribonucleopeptide World

A highly complex RNA world, as is sometimes presented in view of the widespread and diversified use of RNA enzymes, would have encountered many difficulties in passing to a world with catalysis mediated by proteins. These difficulties can be overcome by postulating a very early relationship between the nucleotide and the amino acid components. In particular, after asserting that some characteristics expressed by (nucleotide) coenzymes in catalysis are easier to understand if a close and early relationship between these coenzymes and amino acids is hypothesized, a model is presented for the origin of the enzyme–coenzyme complex. This model is essentially based on an intermediate formed by a tRNA-like molecule covalently linked to a polypeptide. The model attributes the majority of the catalytic role in the ribonucleoprotein world to the latter complex and thus it takes into account the birth of the key intermediate in the origin of protein synthesis—namely, peptidyl-tRNA, which would have otherwise been extremely difficult to select. The predictions of the model are discussed along with its robustness, using the data derived from the study of intermediary metabolism and those from molecular biology. Finally, the appearance of the genetic code in the late phase of the ribonucleopeptide world is discussed. Received: 13 January 1997 / Accepted: 25 July 1997     

Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins

Partition coefficients in poly(ethylene glycol)/dextran aqueous two-phase systems are reported for mixed-casein and its components, alpha, beta and kappa casein. Rates of casein proteolysis by alpha-chymotrypsin and by trypsin are reported in single-phase and aqueous two-phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Contribution of Val/Ile87 residue in the extracellular domain in agonist-induced current responses of the human and rat P2X7 receptors

Purinergic Signalling - The P2X7 receptor (P2X7R) is an ATP-gated cation channel with a critical role in many physiological and pathological processes, and shows prominent functional differences...     

Continuous Flow Routes toward Designer Metal Nanocatalysts

Mono‐ and multimetallic nanoparticles (NPs) have diverse and tunable physicochemical properties that arise from their compositions as well as crystallite size and shape. The ability to control precisely the composition and structure of NPs through synthesis is central to achieving state‐of‐the‐art designer metal NPs for use as catalysts and electrocatalysts. However, a major limitation to the use of designer metal NPs as catalysts is the ability to scale their syntheses while maintaining structural precision. To address this challenge, continuous flow routes to metal NPs involving the use of droplet microreactors are being developed, providing the synthetic versatility necessary to achieve known and completely new nanostructures. This progress report outlines how the chemistry and process parameters of droplet microreactors can be used to achieve high performing nanocatalysts through control of NP composition, size, shape, and architecture and outlines directions toward previously unimaginable nanostructures.     

Graphitic‐Shell Encapsulation of Metal Electrocatalysts for Oxygen Evolution,Oxygen Reduction,and Hydrogen Evolution in Alkaline Solution

Developing highly efficient, cost effective, and environmentally friendly electrocatalysts for the oxygen evolution reaction (OER), oxygen reduction reaction (ORR), and hydrogen evolution reaction (HER) is of interest for sustainable and clean energy technologies, including metal–air batteries and fuel cells. In this work, the screening of electrocatalytic activities of a series of single metallic iron, cobalt, and nickel nanoparticles and their binary and ternary alloys encapsulated in a graphitic carbon shell toward the OER, ORR, and HER in alkaline media is reported. Synthesis of these compounds proceeds by a two‐step sol–gel and carbothermal reduction procedure. Various ex situ characterizations show that with harsh electrochemical activation, the graphitic shell undergoes an electrochemical exfoliation. The modified electronic properties of the remaining graphene layers prevent their exfoliation, protect the bulk of the metallic cores, and participate in the electrocatalysis. The amount of near‐surface, higher‐oxidation‐state metals in the as‐prepared samples increases with electrochemical cycling, indicating that some metallic nanoparticles are not adequately encased within the graphite shell. Such surface oxide species provide secondary active sites for the electrocatalytic activities. The Ni–Fe binary system gives the most promising results for the OER, and the Co–Fe binary system shows the most promise for the ORR and HER.