Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms.

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox interactions in cytochrome c oxidase: from the "neoclassical" toward "modern" models.

Because of recent experimental data on the redox characteristics of cytochrome c oxidase and renewed interest in the role of cooperativity in energy coupling, the question of redox cooperativity in cytochrome c oxidase is reexamined. Extensive redox cooperativity between more than two redox centers, some of which are spectrally invisible, may be expected for this electron transfer coupled proton pump. Such cooperativity, however, cannot be revealed by the traditional potentiometric experiments based on a difference in absorbance between two wavelengths. Multiwavelength analyses utilizing singular value decomposition and second derivatives of absorbance vs. wavelength have revealed a stronger cooperativity than consistent with the "neoclassical" model, which allowed only for weak negative cooperativity between two equipotential one-electron centers. A thermodynamic analysis of redox cooperativity is developed, which includes the possibilities of strong cooperative redox interactions, the involvement of invisible redox centers, conformational changes, and monomer/dimer equilibrations. The experimental observation of an oxidation of one of the cytochromes (a3) with a decrease in applied redox potential is shown to require both strong negative cooperativity and the participation of more than two one-electron centers. A number of "modern" models are developed using the analytical approaches described in this paper. By testing with experimental data, some of these models are falsified, whereas some are retained with suggestions for further testing.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

CULTURE OF HUMAN GENITAL "T-STRAIN" PLEUROPNEUMONIA-LIKE ORGANISMS.

Ford, Denys K. (University of British Columbia, Vancouver, Canada). Culture of human genital "T-strain" pleuropneumonia-like organisms. J. Bacteriol. 84:1028-1034. 1962.-The conditions under which "T-strain" pleuropneumonia-like organisms, as described by Shepard, are best cultured were investigated. The organisms were found to grow on several types of nutrient agar and broth, of which PPLO medium supplemented with yeast extract and horse serum was the simplest. Subculture was possible through broth cultures, provided the broths were not incubated longer than 16 hr. The organisms on agar required either Fortner's anaerobic atmosphere or 10% CO(2), but broth cultures grew aerobically. "T-strains" grew over a pH range of 6.8 to 7.8, and a temperature range of 30 to 36 C. They were viable after storage for 16 days at 4 C and for 90 days at -20 C, and they resisted lyophilization. They were sensitive to 1.5 mug per ml of tetracycline and streptomycin, but were resistant to ampicillin and penicillin. Quantitative studies showed maximal concentration in broth of 10(6) to 10(7) organisms per ml, and logarithmic multiplication for the first 12 hr of broth culture, with a subsequent rapid decline in number. Colonial morphology was maintained after numerous subcultures.     

Protonated polynucleotides structures - 23. The acid-base hysteresis of poly(dG).poly(dC).

The large hysteresis observed during the acid-base titration of poly(dG). poly (dC) was studied by CD and potentiometric scanning curves. Intermediate scanning loops as well as the equilibrium and metastable branches of the hysteresis loop have been determined. The potentiometric titrations showed, however, that the various complexes were not discrete entities, but were linked in "polycomplexes" as had been already suggested. This prevented a thermodynamic study of the system. The acid-base titration was further investigated as a function of ionic strength and temperature. The pK's showed considerably lower ionic strength dependence than observed for polyribonucleotide complexes. The thermal transitions permitted to establish the relative stabilities of the various complexes between pH 2.5 and pH 12.0.     

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic study of the formation of adenine nucleotide · manganese complexes: I. “pH Stat” titration method results

The thermodynamics associated with the Mn^2? · ATP, Mn · ADP^? and Mn · AMP complex formation reactions determined from K potentiometric measurements at I = 0.2 are reported for the temperature range 1–45°C. The K values increase with the lenght of the phosphate chain and with temperature. The limits and the best conditions for use of the “pH stat” titration method are discussed. Comparison with the results obtained by potentiometric and calorimetric methods in the case of Mg · nucleotide complexes is made.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

The monovalent cation "leak" transport in human erythrocytes: an electroneutral exchange process.

The mechanism of the "ground permeability" of the human erythrocyte membrane for K+ and Na+ was investigated with respect to a possible involvement of a previously unidentified specific transport pathway, because earlier studies showed that it cannot be explained on the basis of simple electrodiffusion. In particular, we analyzed and described the increase in the (ouabain+bumetanide+EGTA)-insensitive unidirectional K+ and Na+ influxes as well as effluxes (defined as "leak" fluxes) observed in erythrocytes suspended in low-ionic-strength media. Using a carrier-type model and taking into account the influence of the ionic strength on the outer surface potential according to the Gouy-Chapman theory (i.e., the ion concentration near the membrane surface), we are able to describe the altered "leak" fluxes as an electroneutral process. In addition, we can show indirectly that this electroneutral flux is due to an exchange of monovalent cations with protons. This pathway is different from the amiloride-sensitive Na+/H+ exchanger present in the human red blood cell membrane and can be characterized as a K+(Na+)/H+ exchanger.     

Calorimetric study of crystal growth of ice in hydrated methemoglobin and of redistribution of the water clusters formed on melting the ice.

Calorimetric studies of the melting patterns of ice in hydrated methemoglobin powders containing between 0.43 and 0.58 (g water)/(g protein), and of their dependence on annealing at subzero temperatures and on isothermal treatment at ambient temperature are reported. Cooling rates were varied between approximately 1500 and 5 K min-1 and heating rate was 30 K min-1. Recrystallization of ice during annealing is observed at T > 228 K. The melting patterns of annealed samples are characteristically different from those of unannealed samples by the shifting of the melting temperature of the recrystallized ice fraction to higher temperatures toward the value of "bulk" ice. The "large" ice crystals formed during recrystallization melt on heating into "large" clusters of water whose redistribution and apparent equilibration is followed as a function of time and/or temperature by comparison with melting endotherms. We have also studied the effect of cooling rate on the melting pattern of ice with a methemoglobin sample containing 0.50 (g water)/(g protein), and we surmise that for this hydration cooling at rates of > or = approximately 150 K min-1 preserves on the whole the distribution of water molecules present at ambient temperature.     

Characterization of esterases from Cucurbita pepo cv. "Eskandrani"

Two of the six esterases identified in Cucurbita pepo cv. "Eskandrani" were purified to homogeneity using two chromatography steps: anion exchange and gel filtration. The molecular weights of C. pepo esterases EIc and EII were 50,000 +/- 1500 and 68,000 +/- 1900 Da from gel filtration and 47,000 and 66,000 Da from SDS/PAGE, respectively, suggesting a monomeric structure for both enzymes. Esterases EIc and EII had K(m) values of 1.22 and 1.56 mM and pH optima at 9.0 and 8.0, respectively. The substrate specificity of C. pepo esterases EIc and EII were determined for a number of p-nitrophenyl esters, where their affinity toward these substrates were decreased as carbon atom number increased. Esterases EIc and EII had the same temperature optima, 40 degrees C. Thermal stability studies of esterases EIc and EII indicated that half maximal activities of EIc and EII esterases were reached at 55 degrees C and 50 degrees C, while they lost 45%, 51% and 70%, 77% of their activities after 30 and 90 min of incubation at 40 degrees C, respectively. The effect of different metal cations and inhibitors were examined. The inhibition studies revealed that the active sites of the two esterases contain serine and cysteine residues. The characteristics of C. pepo esterases are closely similar to those of microbial esterases used in food processing and food industry.     

Inframolecular studies of the protonation of adenophostin A: comparison with 1-D-myo-inositol 1,4,5-trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the D-myo-inositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic protonation process of this compound by performing (31)P NMR, (1)H NMR, and potentiometric titration experiments. The logarithms of the first to the fourth stepwise protonation constants are, respectively, log K(1) = 8.48, log K(2) = 6.20, log K(3) = 4.96, and log K(4) = 3.80. The latter constant refers to the protonation equilibrium involving the N1 adenine nitrogen. From the microconstants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophostin A at near physiological pH is very similar to those of inositol 1,4,5-trisphosphate, indicating that differences in phosphate charge cannot account for the high potency of this molecule. The analysis of the (1)H chemical shifts vs pH provided complementary conformational information. In particular, a slight "wrongway shift" of H1" can be related to the protonation of P2, thus indicating a short H1"-P2 distance. Our results are in line with a recently published model in which, however, a certain degree of constraint would keep the ribose 2'-phosphate moiety close to the glucose ring phosphates.     

“秦油2号”油菜植株内含物与桃蚜种群消长的关系

通过对N、P、K不同施肥条件下的“秦油2号”油菜植株内含物与桃蚜种群消长关系的研究与分析,建立了9个数学模型,结果表明可溶性糖含量、含K量、含水量对桃蚜种群消长影响最大;低N、低K、中P组合对桃蚜种群增长不利。     

Buoyant and potentiometric titrations of synthetic polypeptides. I. Six ionizable homopolypeptides in CsCl solutions

The buoyant density and potentiometric titrations of six ionizable homopolypeptides in concentrated CsCI solutions have been studied. These six homopolypeptides were chosen as models of the behavior of ionizable residues in proteins. Their buoyant and potentiometric results will be of value in interpreting the buoyant and potentiometric results observed for proteins. The buoyant densities for all six homopolypeptides were found to increase sigmoidally as the pH is increased. These density changes are interpreted in terms of changes in the hydrations and ion binding which are associated with the titration of the residues. Preferential hydrations for the homopolypeptides are calculated. The buoyant density titrations are combined with the potentiometric titrations to determine the relationship between the buoyant density and the degree of ionization. A better method of computing buoyant densities of proteins is described. The slope of β(ρ) has been computed for CsCl using least-squares curve fitting and this is used in calculating the isoconcentration position. This method has been found to be more accurate than calculating the isoconcentration position from the normalized isoconcentration ratio, which is known only under limited conditions.     

Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example.

A direct method is proposed for obtaining thermodynamic standard functions for native and denatured proteins using experimental data from scanning calorimetry, isothermal calorimetry and potentiometric titrations. The possibility of this approach is demonstrated on the example of lysozyme in the range of pH 1.5-7.0 and temperature 0-100 degrees C. Tests for the validity of the obtained functions of enthalpy and entropy are presented in the form of cyclic processes using experimental data obtained from thermodynamically different pathways. The Gibbs function is checked by comparison with results of an independent method. The methodic problems in determining and checking standard functions for proteins are discussed in detail.     

Two mechanisms by which fluorescent oxonols indicate membrane potential in human red blood cells.

Optical potentiometric indicators have been used to monitor the transmembrane electrical potential (Em) of many cells and organelles. A better understanding of the mechanisms of dye response is needed for the design of dyes with improved responses and for unambiguous interpretation of experimental results. This paper describes the responses to delta Em of 20 impermeant oxonols in human red blood cells. Most of the oxonols interacted with valinomycin, but not with gramicidin. The fluorescence of 15 oxonols decreased with hyperpolarization, consistent with an "on-off" mechanism, whereas five oxonols unexpectedly showed potential-dependent increases in fluorescence at less than 2 microM [dye]. Binding curves were determined for two dyes (WW781, negative response and RGA451, positive response) at 1 mM [K]o (membrane hyperpolarized with gramicidin) and at 90 mM [K]o (delta Em = 0 with gramicidin). Both dyes showed potential-dependent decreases in binding. Changes in the fluorescence of cell suspensions correlated with changes in [dye]bound for WW781, in accordance with the "on-off" mechanism, but not for RGA451. Large positive fluorescence changes (greater than 30%) dependent on Em were observed between 0.1 and 1.0 microM RGA451. A model is suggested in which RGA451 moves between two states of different quantum efficiencies within the membrane.     

Mixed Chelates of Ca(II)-Pyridine-2,6-Dicarboxylate with Some Amino Acids Related to Bacterial Spores

The high resistance of bacterial spores to heat has been repeatedly postulated to be due to stabilization of spore biopolymers by metal chelate compounds. Binding of calcium dipicolinic acid (Ca(II)-DPA) with spore proteins and amino acids has been discussed in the literature, but equilibrium data are generally lacking. By means of potentiometric pH titrations at 25 degrees C and an ionic strength of 1.0 (KNO(3)), the formation of Ca(II)-DPA (1:1 and 1:2) chelates and the interactions of Ca(II)-DPA chelate with a mole of each of three typical amino acids viz., cysteine, alanine, and glycine has been investigated. Analysis of the potentiometric data indicates that calcium and DPA forms 1:1 and 1:2 chelates with log K(ML1) = 4.39 +/- 0.01 and log K(ML2) = 2.25 +/- 0.01. In the presence of an equimolar amount of each of the amino acids under consideration, the Ca(II)-DPA chelate forms mixed ligand (ternary) chelate yielding the following stepwise stability constants: log K(1) = 4.17 +/- 0.01, log K(2) = 0.78 +/- 0.01 for cysteine, log K(1) = 4.06 +/- 0.01, log K(2) = 0.65 +/- 0.01 for alanine, and log K(1) = 4.30 +/- 0.02, log K(2) = 0.11 +/- 0.01 for glycine. Methods for calculating the stability constants of the mixed ligand system have been developed. On the basis of the potentiometric equilibrium data, possible structures for the various calcium chelate species are discussed. The data suggest that the differences in heat resistance of various strains of bacterial spores may conceivably be related to the differences in composition and stability of coordination complexes in the spore.     

Thermodynamic binding parameters of individual epitopes of multivalent carbohydrates to concanavalin a as determined by "reverse" isothermal titration microcalorimetry.

The preceding paper [Dam, T. K., Roy, R., Pagé, D., and Brewer, C. F. (2002) Biochemistry 41, 1351-1358] demonstrated that Hill plots of isothermal titration microcalorimetry (ITC) data for the binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to the lectin concanavalin A (ConA) show increasing negative cooperativity upon binding of the analogues to the lectin. The present study demonstrates "reverse" ITC experiments in which the lectin is titrated into solutions of di- and trivalent analogues. The results provide direct determinations of the thermodynamics of binding of ConA to the individual epitopes of the two multivalent analogues. The n values (number of binding sites per carbohydrate molecule) derived from reverse ITC demonstrate two functional binding epitopes on both the di- and trivalent analogues, confirming previous "normal" ITC results with the two carbohydrates [Dam, T. K., Roy, R., Das, S. K., Oscarson, S., and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223-14230]. The reverse ITC measurements show an 18-fold greater microscopic affinity constant of ConA for the first epitope of the divalent analogue versus its second epitope and a 53-fold greater microscopic affinity constant of ConA binding to the first epitope of the trivalent analogue versus its second epitope. The data also demonstrate that the microscopic enthalpies of binding of the two epitopes on the di- and trivalent analogues are essentially the same and that differences in the microscopic K(a) values of the epitopes are due to their different microscopic entropies of binding values. These findings are consistent with the increasing negative Hill coefficients of these analogues binding to ConA in the previous paper.