A proposed solution for the determination of the ionization constants of sets of ionizinggroups in proteins

A mixture of acids with known pK′ and in a known concentration was dissolved to simulate the ionic character of hemoglobin. The titration curves of the mixture were obtained in water and 3.6 M KCl at 20°C. These curves were subjected to analysis by a reiterative curve-fitting procedure to determine if one could evaluate both the group ionization constants and the numbers of groups. This approach was successful in that the calculated parameters were within the experimental error encountered in obtaining these constants on each of the model compounds. However, analysis for the electrostatic interaction parameter w indicated that there was a possible effect on the ionization of formic acid, pyridine, imidazole, and the α-amino group of glycylglycine in water and on imidazole and the α-amino group of glycylglycine in 3.6 M KCl.     

The role of formate for the growth of Rhodococcus opacus UFZ B 408 on pyridine

R. opacus UFZ B 408 is able to use pyridine, a potentially growth-inhibiting substrate, as the sole source of carbon, energy and nitrogen. In a previous publication [1] we reported that with the simultaneous utilization of a second carbon and energy source in carbon-substrate-limited chemostat culture, stable steady states could be achieved at higher dilution rates than with growth on pyridine as the sole substrate. Owing to the higher growth yield during growth on such a substrate mixture, both the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone. Therefore, the critical growth-inhibitory pyridine concentration was only achieved at a higher dilution rate. With the investigations presented here in carbon-substrate-limited continuous culture, the simultaneous utilization of pyridine and formate by R. opacus UFZ B 408 was studied. The yield coefficient during growth on pyridine as the sole substrate amounted to about 0.55 g dry mass/g pyridine. Theoretically, however, the carbon-metabolism-determined yield coefficient should have been about 0.915 g dry mass/g pyridine. Because of the difference between these two values the conclusion was drawn that pyridine is energetically deficient. That means that during growth on pyridine a part of the substrate was dissimilated to supply the energy required for the incorporation of the pyridine carbon into biomass. Formate cannot be used as a carbon source for growth by R. opacus UFZ B 408. However, with growth on pyridine, formate was oxidized simultaneously. During growth on pyridine/formate mixtures, the yield coefficient could be enhanced up to 0.7 g dry mass/g pyridine. That means that biologically usable energy, generated in the course of the formate oxidation, was used for the assimilation of pyridine carbon. The increase in the yield coefficient was related to the utilization ratio of formate to pyridine in a linear manner. However, the carbon-metabolism-determined yield coefficient of 0.915 g dry mass/g pyridine could not be achieved. That can be put down to the fact that R. opacus UFZ B 408 possesses only a limited capacity to oxidize externally supplied formate. Because of the limited formate oxidation capacity the probability is low that, with simultaneous utilization of formate, stable steady states could be achieved at substantially higher dilution rates than with growth on pyridine alone. Enzymatic studies revealed the induction of both NAD(P)⁺-linked glutaric dialdehyde dehydrogenase and isocitrate lyase during growth on pyridine. Therefore, the conclusion was drawn that pyridine is metabolized by R. opacus UFZ B 408 via the same pathway described for the utilization of pyridine by Nocardia Z1 [2]. This conclusion implies that the ability to oxidize formate represents a metabolic performance which seems not to be directly related to the pyridine metabolism of R. opacus UFZ B 408.     

Simultaneous biodegradation of pyridine and quinoline by two mixed bacterial strains

Experiments were conducted to provide data on the effectiveness of bioaugmentation in the removal of pyridine and quinoline from different wastewaters. A pyridine-degrading bacterial strain (Paracoccus sp. BW001) and a quinoline-degrading strain (Pseudomonas sp. BW003) were isolated from the activated sludge of a coking wastewater treatment plant. In this study, a consortium of these two bacterial strains was used as inoculum to simultaneously degrade pyridine and quinoline in three types of wastewaters: sterile synthetic, domestic, and industrial. In addition, variation of the bacterial community structures during degradation was monitored by denaturing gradient gel electrophoresis and amplicon length heterogeneity polymerase chain reaction techniques. The results of our experiments indicate that pyridine and quinoline can be removed efficiently using this inoculum but that the degradation process results in the production of ammonium as a by-product. Also, in the two actual wastewaters investigated, we observed that several autochthonous strains of bacteria in both the domestic and industrial wastewater were tolerant of pyridine and quinoline and grew rapidly.     

Electrophysiological studies of pyridine-sensitive units on the crayfish walking leg

Summary Pyridine-sensitive units located on the walking legs of the crayfishAustropotamobius torrentium were studied by extracellular recording of the action potentials of single afferent fibers. To characterize the sensitivity and specificity of the pyridine receptor, 79 pyridine analogs and other related substances were tested on 70 neurons. The maximum impulse frequency of the response was used to construct dose-response curves. The effectiveness of stimulatory substances was characterized at the half-maximal-response frequency, K_M. The effectiveness rank order of the substances was found to be the same for all units tested. The most effective substances were: pyrazinecarboxamide > 3-acetylpyridine > nicotinamide > pyridine-3-aldoxime, with K_M values of 1.5×10^–6, 4× 10^–6, 10^–5 and 4 x 10^–5 mol/1, respectively. The inferred structural requirements for an optimal stimulatory molecule are that it have a N-containing aromatic ring system with a specific substituent in them position.     

Chiral Amine Enantiomeric Excess Determination Using Self‐Assembled Octahedral Fe(II)‐Imine Complexes

A receptor assembly composed of iron(II) triflate and pyridine‐2,6‐dicarbaldehyde was used to determine the enantiomeric excess (ee) of alpha‐chiral primary amines using circular dichroism spectroscopy. The alpha chiral amines condense with the dialdehyde to form a diimine, which forms a 2:1 octahedral complex with iron(II). The ee values of unknown concentrations of alpha‐chiral amines were determined by constructing calibration curves for each amine and then measuring the ellipticity at 600 nm. This improves our previously reported assay for ee determination of chiral primary amines by further increasing the wavelength at which CD is measured and reducing the absolute error of the assay. Chirality 27:294–298, 2015. 2015 Wiley Periodicals, Inc.     

Effect of pyridine homologues on the basal rate of electron transport and H+/e − ratio in chloroplasts

Low concentrations of hydrophobic pyridine homologues (1 mM) were found to increase the rate of the Hill reaction in chloroplasts without significantly affecting either the steady-state proton uptake or the rate of proton leakage in the dark. By assuming that the organic base can be bound to two types of independent binding sites in the thylakoid membrane with dissociation constantsK ₁ andK ₂ respectively, the kinetic data can be treated quantitatively. The values ofK ₁ andK ₂ determined by the treatment are in the same relative order as the hydrophobicities of the pyridine homologues:K ₁=1.16 mM andK ₂=54 mM for pyridine; 0.6 and 38 mM for 4-picoline; 0.27 and 31 mM for 4-ethylpyridine, 0.10 and 4.2 mM for 4-t-butylpyridine; 0.08 and 3.2 mM for 4-n-butylpyridine. The rates of oxygen generation and proton uptake by illuminated chloroplasts with either ferricyanide or 1,4-benzoquinone as the electron acceptor were also measured in the presence of various pyridine homologues. Low concentration of pyridine homologues were found to decrease the H⁺/e ^– ratio. This last observation seems to substantiate an indirect coupling mechanism between electron transport and proton translocation.Abbreviations Chl chlorophyll - CF₀ - CF₁ the coupling factor complex of chloroplast - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Tricine N-tris-(hydroxymethyl)methylglycine     

Simultaneous utilization of pyridine and fructose by Rhodococcus opacus UFZ B 408 without an external nitrogen source

 A bacterium classified as Rhodococcus opacus, which is able to use pyridine (a potentially growth-inhibiting substrate) as its sole source of carbon, energy and nitrogen, was isolated. In a carbon-limited chemostat culture, the kinetics was determined for growth on both pyridine and a mixture of pyridine and fructose (9 mM/22.15 mM). With growth on pyridine, stable steady states were achieved up to dilution rates of about 0.1 h^-1. A further increase in the dilution rate resulted in the progressive accumulation of pyridine in the culture liquid and the cells were washed out. The maximum specific growth rate (μ_max = 0.23 h^-1) and the K _S value (0.22 mM) for growth on pyridine were determined from the residual pyridine concentrations measured within the range of stable steady states. With growth on the substrate mixture, the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone, and stable steady states were established at dilution rates of up to 0.13 h^-1. The maximum pyridine degradation rate was enhanced to 270 mg pyridine l^-1 h^-1 compared to 210 mg pyridine l^-1 h^-1with growth on pyridine as a single substrate. An external nitrogen source did not need to be added in the case of growth on the substrate mixture. Fructose was assimilated by means of ammonium released from pyridine. Analysis of the nitrogen balance furnished proof that pyridine is an energy-deficient substrate; pyridine was assimilated and dissimilated at a ratio of 1 mol/0.67 mol respectively. The resulting yield coefficient was about 0.55 g dry weight/g pyridine. Moreover, it was demonstrated that, in regard to the biologically usable energy, 1 mol pyridine corresponds to 0.43 mol fructose. Received: 3 July 1995/Received revision: 19 October 1995/Accepted: 24 October 1995     

Microbial degradation of pyridine and α-picoline using a strain of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Nocardia</Emphasis> sp.

Biodegradation of pyridine and α-picoline (2-methyl pyridine) by Pseudomonas pseudoalcaligenes-KPN and Nocardia sp. isolated from garden soil were investigated in batch culture experiments. Pyridine and α-picoline (50–200 mg L⁻¹) were used as sole source of carbon and energy in the investigation. The kinetic constants were evaluated for pyridine and α-picoline degradation under optimized nutritional (C, N, P) and environmental (pH, temperature) conditions. The values of bio-kinetic constant obtained in the present investigation indicate the usefulness of both the cultures for treatment of waste containing pyridine and its derivatives.     

Isolation and characterization of potential aerobic bacteria capable for pyridine degradation in presence of picoline,phenol and formaldehyde as co-pollutants

Pyridine, heterocyclic aromatic compound is known to be toxic, carcinogenic and teratogenic to several living organisms. In this study, two aerobic bacteria ITRCEM1 and ITRCEM2 capable for pyridine degradation were isolated and characterized as Bacillus cereus (DQ435020) and Alcaligenes faecalis (DQ435021), respectively. For pyridine degradation, mixed bacterial culture was found more effective compared to axenic culture ITRCEM1 and ITRCEM2 degrading 94.23, 67.84, and 83.35% pyridine, respectively, at 144 h incubation period at pH 7.0 ± 0.1, temp 37 ± 2°C and shaking rate 125 rpm in MSM containing 1% glucose and 0.2% peptone as carbon and nitrogen source, respectively. The presence of phenol and formaldehyde in MSM has shown inhibitory effect on pyridine degradation whereas picoline has favored the bacterial growths and pyridine degradation. Further, the HPLC analysis has shown the reduction in peaks compared to controls, indicating that reduction in peak area might be largely attributed to the bacterial degradation of pyridine by bacterial catabolic enzymes.     

The relationship between ATP and an electrogenic pump in the plasma membrane ofNeurospora crassa

Summary Sudden respiratory blockade has been used to study rapid changes of the resting membrane potential, of intracellular adenosine 5-triphosphate (ATP) levels, and of pyridine nucleotide reduction inNeurospora crassa. Membrane depolarization occurs with a first-order rate constant of 0.167 sec^–1, following a lag period of about 4 sec, at 24°C (ambient temperature). This depolarization is several-fold too slow to be directly linked to electron transfer, as judged from the rate of pyridine nucleotide reduction, but has essentially the same rate constant as the decay of ATP. The latter process, however, shows no lag period after the respiratory inhibitor is introduced. Plots of membrane potential versus the intracellular ATP concentration yield saturation curves which are readily fitted by a Michaelis equation, to which is added a constant term representing the diffusion component of membrane potential. Parameters obtained from such fits indicate the maximal voltage which the pump can develop at high ATP levels to be 300 to 350 mV, with an apparentK _1/2 of 2.0mm. The data strongly suggest that an electrogenic ion pump in the plasma membrane ofNeurospora is fueled by ATP; comparison of the measured membrane potentials with the energy available from hydrolysis of ATP indicates that two ions could be pumped for each molecule of ATP split.     

Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries

The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.     

Influence of a supplementary carbon source on biodegradation of pyridine by freely suspended and immobilizedPimelobacter sp.

The effect of the presence of supplementary glucose or acetate on the growth and pyridine-degrading activity of freely suspended and calcium-alginate-immobilizedPimelobacter sp. was investigated. Although the supplementary carbon sources could be degraded simultaneously with pyridine,Pimelobacter sp. exhibited a preference for pyridine over supplementary carbon sources. Thus, the pyridine-degrading activity of the freely suspended cells was not decreased significantly by the addition of either glucose (1.5–6 mM) or acetate (6–24 mM) to the pyridine (6–24 mM). In the semi-continuous immobilized cell culture, immobilized cells also exhibited a preference for pyridine over supplementary carbon sources and did not switch their substrate preference throughout the culture. Owing to a high cell concentration, the volumetric pyridine degradation rate at 24 mM pyridine in the immobilized cell culture was approximately six times higher than that in the freely suspended cell culture. Furthermore, the immobilized cells could be reused 16 times without losing their pyridine-degrading activity during the culture period tested. Taken together, the use of immobilizedPimelobacter sp. for the degradation of pyridine is quite feasible because of the preference for pyridine over supplementary carbon sources, the high volumetric pyridine degradation rate, and the reusability of immobilized cells.     

The Synthesis of Pyrazol0[3,4-c]Pyridine C-Nucleosides

Abstract

The synthesis of two members of the pyrazolo[3,4-c]pyridine C-nucleoside family is accomplished utilizing the 1.3-dipolar cycloaddition reaction of a diazo-ribofuranosyl derivative and an appropriate allenic diester.     

Microbial degradation and metabolic pathway of pyridine by a <Emphasis Type="Italic">Paracoccus</Emphasis> sp. strain BW001

A bacterial strain using pyridine as sole carbon, nitrogen and energy source was isolated from the activated sludge of a coking wastewater treatment plant. By means of morphologic observation, physiological characteristics study and 16S rRNA gene sequence analysis, the strain was identified as the species of Paracoccus. The strain could degrade 2,614 mg l⁻¹ of pyridine completely within 49.5 h. Experiment designed to track the metabolic pathway showed that pyridine ring was cleaved between the C₂ and N, then the mineralization of the carbonous intermediate products may comply with the early proposed pathway and the transformation of the nitrogen may proceed on a new pathway of simultaneous heterotrophic nitrification and aerobic denitrification. During the degradation, NH₃-N occurred and increased along with the decrease of pyridine in the solution; but the total nitrogen decreased steadily and equaled to the quantity of NH₃-N when pyridine was degraded completely. Adding glucose into the medium as the extra carbon source would expedite the biodegradation of pyridine and the transformation of the nitrogen. The fragments of nirS gene and nosZ gene were amplified which implied that the BW001 had the potential abilities to reduce NO₂ ⁻ to NO and/or N₂O, and then to N₂.     

15N-NMR spectroscopy. 34. Stereospecificity of the polymerization of D,L-leucine N-carboxyanhydride and γ-OMe-D,L-glutamic acid N-carboxyanhydride

D ,L -Leucine-N-carboxyanhydride (D ,L -Leu-NCA) and γ-methyl-D ,L -glutamic acid N-carboxyanhydride (γ-OMe-D ,L -Glu-NCA) were synthesized with ca. 2.5% ¹⁵N enrichment. Their polymerizations were conducted under a variety of conditions using benzylamine, triethylamine potassium tert-butanolate, and pyridine as initiators. The 40.55-MHz ¹⁵N-nmr spectra of the resulting stereocopolypeptides measured in trifluoroacetic acid display at least four signals, representing the isotatic, syndiotactic, and two heterotactic triads. From the signal intensities it was concluded that these NCAs behave nearly identically. With benzylamine initiation the formation of isotactic blocks is slightly favored, and they are still more predominant when strong bases are used as initiators. Initiation by pyridine favors the formation of syndiotactic sequences. However, in all cases the average lengths of the stereoblocks never exceeded 4 monomer units. The low stereospecificity of most polymerizations of D ,L -NCAs is confirmed by the high degree of solubility of the resulting poly(D ,L -amino acids) in aprotic solvents. Penultimate effects are weak or absent, so that most polymerizations follow Bernoullian type statistics. Deviations from these statistics were found for polymerizations in pyridine.     

Pyridine alkaloid distribution in the hoplonemertines

Hoplonemertines possess a family of pyridine compounds affecting the nervous system (Kem, 1985). Anabaseine, the first pyridine to be isolated, stimulates nicotinic receptors. Two other substances, 2,3-bipyridyl and nemertelline (a tetrapyridyl) were isolated from Amphiporus angulatus. In this study samples of 19 species of hoplonemertines were surveyed for the presence of stable pyridines using thin layer chromatography. Pyridines were selectively detected with the Konig reagent.Pyridines were found to be nearly ubiquitous constituents of this taxonomic group. Nevertheless, individual species often differed in the pyridines present as well as the variety of compounds present. A new Konig-reactive pyridine was found in 11 hoplonemertine species. Only Zygonemertes virescens and Amphiporus lactifloreus contained anabasine. Only A. angulatus contained 2,3-bipyridyl and nemertelline. This initial survey suggests that differences in pyridine compositions between hoplonemertines may be a useful taxonomic character.     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. III. Characterization of the ATP formation onset lag and post-illumination phosphorylation for thylakoids exhibiting localized or bulk-phase delocalized energy coupling

When 100 mM KCl replaced sucrose in a chloroplast thylakoid stock suspension buffer, the membranes were converted from a localized proton gradient to a delocalized proton gradient energy coupling mode. The KCl-suspended but not the sucrose-suspended thylakoids showed pyridine-dependent extensions of the ATP onset lag and pyridine effects on post-illumination phosphorylation. The ATP formation assays were performed in a medium of identical composition, using about a 200-fold dilution of the stock thylakoid suspension; hence the different responses were due to the pretreatment, and not the conditions present in the phosphorylation assay. Such permeable buffer effects on ATP formation provide a clear indicator of delocalized proton gradients as the driving force for phosphorylation. The pyridine-dependent increases in the onset lags (and effects on post-illumination phosphorylation) were not due to different ionic conductivities of the membranes (measured by the 515 nm electrochromic absorption change), H⁺/e ^– ratios, or electron transport capacities for the two thylakoid preparations. Thylakoid volumes and [ 14C]pyridine equilibration were similar with both preparations. The KCl-induced shift toward a bulk-phase delocalized energy coupling mode was reversed when the thylakoids were placed back in a low-salt medium.Proton uptake, at the ATP-formation energization threshold flash number, was much larger in the KCl-treated thylakoids and they also had a longer ATP formation onset lag, when no pyridine was present. These results are consistent with the salt treatment exposing additional endogenous buffering groups for interaction with the proton gradient. The concomitant appearance of the pyridine buffer effects implies that the additional endogenous buffering groups must be located on proteins directly exposed in the aqueous lumen phase.Kinetic analysis of the decay of the post-illumination phosphorylation in the two thylakoid preparations showed different apparent first-order rate constants, consistent with there being two different compartments contributing to the proton reservoirs that energize ATP formation. We suggest that the two compartments are a membrane-phase localized compartment operative in the sucrose-treated thylakoids and the bulk lumen phase into which protons readily equilibrate in the KCl-treated thylakoids.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Quantitative structure activity relationships of some pyridine derivatives as corrosion inhibitors of steel in acidic medium

Quantum chemical calculations using the density functional theory (B3LYP/6-31G* DFT) and semi-empirical AM1 methods were performed on ten pyridine derivatives used as corrosion inhibitors for mild steel in acidic medium to determine the relationship between molecular structure and their inhibition efficiencies. Quantum chemical parameters such as total negative charge (TNC) on the molecule, energy of highest occupied molecular orbital (E _HOMO), energy of lowest unoccupied molecular orbital (E _LUMO) and dipole moment (μ) as well as linear solvation energy terms, molecular volume (Vi) and dipolar-polarization (π*) were correlated to corrosion inhibition efficiency of ten pyridine derivatives. A possible correlation between corrosion inhibition efficiencies and structural properties was searched to reduce the number of compounds to be selected for testing from a library of compounds. It was found that theoretical data support the experimental results. The results were used to predict the corrosion inhibition of 24 related pyridine derivatives.