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PubMed1991

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.

Animals↗
PubMed1994

A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without Schiff base deprotonation.

Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.

Aspartic Acid↗
PubMed1996

Interaction of electron leak and proton leak in respiratory chain of mitochondria--proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen.

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, an exogenous superoxide (O2.-) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2.- generating system, it was found that both sources O2.- showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and H+/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondrial inner membrane by succinate oxidation. The injurious effects of O2.- on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. O2.- has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3,4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous O2.- source from ischemia-reperfused heart on proton pumping activity, H+/2e ratio and transmembrane potential of energized mitochondria. The experimental results presented here clearly showed that O2.- radicals induce an increase of proton leak in mitochondria directly related to the molecular mechanism of early phase damage of ischemia-reperfused heart, and also provide evidence to support our recent proposed hypothesis that electron leakage to oxygen to form O2.- in respiratory chain is one of the mechanical cause of proton leakage in mitochondria.

Animals↗
PubMed2003

Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (DeltapH) across the vacuolar membrane, being approximately 2H+/ATP at high DeltapH (4 pH units) and increasing to >4H+/ATP for small or zero DeltapH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit--which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

Adenosine Triphosphate↗
PubMed2000

The proton to electron stoichiometry of steady-state photosynthesis in living plants: A proton-pumping Q cycle is continuously engaged.

A noninvasive technique is introduced with which relative proton to electron stoichiometries (H(+)/e(-) ratios) for photosynthetic electron transfer can be obtained from leaves of living plants under steady-state illumination. Both electron and proton transfer fluxes were estimated by a modification of our previously reported dark-interval relaxation kinetics (DIRK) analysis, in which processes that occur upon rapid shuttering of the actinic light are analyzed. Rates of turnover of linear electron transfer through the cytochrome (cyt) b(6)f complex were estimated by measuring the DIRK signals associated with reduction of cyt f and P(700). The rates of proton pumping through the electron transfer chain and the CF(O)-CF(1) ATP synthase (ATPase) were estimated by measuring the DIRK signals associated with the electrochromic shifting of pigments in the light-harvesting complexes. Electron transfer fluxes were also estimated by analysis of saturation pulse-induced changes in chlorophyll a fluorescence yield. It was shown that the H(+)/e(-) ratio, with respect to both cyt b(6)f complex and photosystem (PS) II turnover, was constant under low to saturating illumination in intact tobacco leaves. Because a H(+)/e(-) ratio of 3 at a low light is generally accepted, we infer that this ratio is maintained under conditions of normal (unstressed) photosynthesis, implying a continuously engaged, proton-pumping Q cycle at the cyt b(6)f complex.

Chlorophyll↗
PubMed1995

Solution structures of the i-motif tetramers of d(TCC), d(5methylCCT) and d(T5methylCC): novel NOE connections between amino protons and sugar protons.

BACKGROUND: At slightly acid or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines form a tetramer structure in which two parallel-stranded duplexes have their hemi-protonated C.C+ base pairs face-to-face and fully intercalated, in a so-called i-motif, first observed serendipitously in [d(TC5)]4. RESULTS: A high-definition structure of [d(TCC)]4 was computed on the basis of inter-residue distances corresponding to 21 NOESY cross-peaks measured at short mixing times. A similarly defined structure of [d(5mCCT)]4 was also obtained. A small number of very characteristic (amino proton)-(sugar proton) cross-peaks entails the intercalation topology. The structure is generally similar to that of [d(TC5)]4. The sequence d(T5mCC) forms two tetramers in comparable proportions. The intercalation topologies are read off the two patterns of (amino proton)-(sugar proton) cross-peaks: one is the same as in the d(TCC) tetramer, the other has the intercalated strands shifted by one base, which avoids the steric hindrance between the methyl groups of the 5mC pairs of the two duplexes. CONCLUSIONS: The structures obtained in this work and the procedures introduced to characterize them and to solve the problems linked to the symmetry of the structure provide tools for further exploring the conditions required for formation of the i-motif.

Base Sequence↗
PubMed2000

Proton affinity of uracil. A computational study of protonation sites.

Relative stabilities of uracil tautomers and cations formed by gas-phase protonation were studied computationally with the B3LYP, MP2, QCISD, and QCISD(T) methods and with basis sets expanding from 6-31G(d,p) to 6-311+G(3df,2p). In accordance with a previous density functional theory study, the dioxo tautomer 1a was the most stable uracil isomer in the gas phase. Gibbs free energy calculations using effective QCISD(T)/6-311+G(3df,2p) energies suggested >99.9% of 1a at equilibrium at 523 K. The most stable ion isomer corresponded to N-1 protonated 2,4-dihydroxypyrimidine, which however is not formed by direct protonation of 1a. The topical proton affinities in 1a followed the order O-8 > O-7 > C-5 > N-3 > N-1. The thermodynamic proton affinity of 1a was calculated as 858 kJ mol(-1) at 298 K. A revision is suggested for the current estimate included in the ion thermochemistry database.

Journal Article↗
PubMed2001

A study of light-induced proton transfer from gas phase (radical) cations to reference bases. Bracketing of proton transfer from excited ions and associated reaction kinetics.

By use of Fourier transform ion cyclotron resonance, it is shown that protonated naphthalene when excited with laser light of 488 nm is more reactive in proton transfer to reference bases than in its ground state. The excitation leads to reaction with bases for which proton transfer in the ground state is endothermic up to a detected maximum of 60 kJ/mol. For indene radical cations excited at 514.5 nm, it is shown that the rate constant for proton transfer to 3-pentanone is either about 10 or about 100 times lower than the rate constant for relaxation by collisions with 3-pentanone. From the energy deposited in the ions, 0.5-0.6 eV is available for proton transfer to a base which seems reasonable when taking into account a complete randomization of the initially deposited energy.

Journal Article↗
PubMed1985

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study.

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.

Adenosine Triphosphate↗
PubMed2002

Synthesis, crystal structure, and H/D exchange of the inside protonated form of the cage imine 4,8,12-triaza-1-azoniatricyclo[6.6.3.2(4,12)]nonadec-1(15)-ene. A model for proton transfer through an aliphatic membrane.

The reaction of the inside protonated form of the tricyclic amine 1,4,8,12-tetraazatricyclo[6.6.3.2(4,12)]nonadecane (1) with iron(III) affords the inside monoprotonated form of the corresponding imine 4,8,12-triaza-1-azoniatricyclo[6.6.3.2(4,12)]nonadec-1(15)-ene (2), which was isolated as the tetrabromozincate salt (2a) in a yield of 78%. The crystal structure of 2a has been solved by X-ray diffraction at T = 120 K. In the imine cation the acidic hydrogen atom and the lone pairs of the nitrogen atoms are oriented toward the inside of the cavity. The acidic hydrogen atom is bound to a nitrogen atom belonging to the triazacyclononane entity. The imine double bond is situated between the N-atom of the triazacyclononane entity and the C-atom belonging to one of the three trimethylene bridges. The imine 2 is stable in acidic solution and the inside coordinated proton is very robust in acidic solution. In basic solution the imine reacts fast to give a quantitative formation of the inside protonated form of the hemiaminal 1,4,8,12-tetraazatricyclo[6.6.3.2(4,12)]nonadecan-5-ol (3). The equilibrium constant K(im) = [3][H(+)]/[2] was determined at three different temperatures from potentiometric measurements, which gave K(im) = 1.57(1) x 10(-5) M at 25 degrees C, Delta S degrees = -83(1) J mol(-1) K(-)(1),and Delta H degrees = 2.6(3) kJ mol(-1) at I = 1.0 M (NaCl). The inside coordinated proton in 3 is labile in basic solution and the rate for NH/ND exchange was determined by (1)H NMR at three different temperatures. The reaction followed the expression k(obs) = k(ex)[OD(-)] with k(ex) = 0.0978(30) dm(3) mol(-1) s(-1) at 25 degrees C, Delta S(++) = 87(4) J mol(-1) K(-1), and Delta H(++) = 104.9(11) kJ mol(-1) at I = 1.0 M (NaCl). The exchange rate is more than 5 x 10(6) times faster than that of the parent saturated cage 1. This extreme enhancement of reactivity is explained by an intramolecular proton transfer reaction mediated by hydroxy and oxy groups flipping in and out of the cavity, which mechanistically has resemblance to the transport of ions in a biological system.

Journal Article↗
PubMed2003

Influence of media and homoconjugate pairing on transition metal hydride protonation. An IR and DFT study on proton transfer to CpRuH(CO)(PCy3).

The interaction of the ruthenium hydride complex CpRuH(CO)(PCy(3)) (1) with proton donors HOR of different strength was studied in hexane and compared with data in dichloromethane. The formation of dihydrogen-bonded complexes (2) and ion pairs stabilized by hydrogen bonds between the dihydrogen ligand and the anion (3) was observed. Kinetics of the interconversion from 2 to 3 was followed at different (CF(3))(3)COH concentrations between 200 and 240 K. The activation enthalpy and entropy values for proton transfer from the dihydrogen-bonded complex 2 to the (eta(2)-H(2))-complex 3 (DeltaH() = 11.0 +/- 0.5 kcal/mol and DeltaS() = -19 +/- 3 eu) were obtained for the first time. The results of the DFT study of the proton transfer process, taking CF(3)COOH and (CF(3))(3)COH as a proton donors and introducing solvent effects in the calculation with the PCM method, are presented. The role of homoconjugate pairs [ROHOR](-) in the protonation is analyzed by means of the inclusion of an additional ROH molecule in the calculations. The formation of the free cationic complex [CpRu(CO)(PCy(3))(eta(2)-H(2))](+) is driven by the formation of the homoconjugated anionic complex [ROHOR](-). Solvent polarity plays a significant role stabilizing the charged species formed in the process. The theoretical study also accounts for the dihydrogen release and production of CpRu(OR)(CO)(PCy(3)), observed at temperatures above 250 K.

Journal Article↗
PubMed2003

Kinetics and mechanism of the proton transfer to CpFe(dppe)H: absence of a direct protonation at the metal site.

The reaction between CpFe(dppe)H and a number of different proton donors (2-fluoroethanol, MFE; 2,2,2-trifluoroethanol, TFE; hexafluoro-2-propanol, HFIP; perfluoro-tert-butyl alcohol, PFTB; and trifluoroacetic acid, TFA) has been investigated spectroscopically by variable-temperature infrared, UV-visible, and NMR spectroscopy, and has been measured kinetically by the stopped-flow technique with UV-visible detection. The low-temperature IR study shows the establishment of hydrogen-bonding interactions which involve the hydride ligand as the proton accepting site. This investigation quantifies the thermodynamics of the hydrogen-bonding interaction and the basicity factor (E(j)) of the hydride complex. All techniques agree in indicating an equilibration process, after the immediate hydrogen-bond formation, between the hydride complex and an intermediate dihydrogen complex, [CpFe(dppe)(H(2))](+). The equilibrium is shifted toward the dihydrogen complex to a greater extent for the stronger alcohols and for higher alcohol/Fe ratios. The observed equilibration rate constant is linearly dependent on the alcohol concentration, in agreement with the involvement of two alcohol molecules and the formation of a homoconjugate pair. The rate constant increases with the acidity of the proton donor (TFE < HFIP < PFTB < TFA). The rate of the subsequent irreversible isomerization leading to the classical dihydride complex, [CpFe(dppe)H(2)](+), is first order, and the rate constant does not depend on the proton donor nature. The reaction continues, if conducted in CH(2)Cl(2), with a third, slower step leading to the paramagnetic [CpFe(dppe)Cl](+) product. The kinetic data are in accord with an isomerization mechanism consisting of an intramolecular reorganization, leading in one step from the dihydrogen complex to the classical dihydride species, and disagree with the occurrence of a proton-transfer process at the metal site.

Journal Article↗
PubMed1984

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.

Carbonic Anhydrases↗
PubMed2000

Short-term treatment with proton pump inhibitors, H2-receptor antagonists and prokinetics for gastro-oesophageal reflux disease-like symptoms and endoscopy negative reflux disease.

BACKGROUND: Heartburn affects 25% of the adult population on a monthly basis and represents the core symptom of gastro-oesophageal reflux disease (GORD). Treatment is readily available and puts a large demand on healthcare budgets. Research in the past has focused largely on the treatment of oesophagitis. A majority of GORD patients show no endoscopic abnormalities and in daily practice most patients are treated empirically. OBJECTIVES: Summarise, quantify and compare the efficacy of the short-term use of proton pump inhibitors (PPI), H2-receptor antagonists (H2RA) and prokinetics in the empirical treatment of GORD and the treatment of endoscopy negative reflux disease (ENRD). SEARCH STRATEGY: Electronic searches were performed of the Cochrane Controlled Trials Register, MEDLINE and EMBASE. Bibliographies were screened. SELECTION CRITERIA: Included were randomised controlled trials focussing on symptomatic outcome after short-term treatment for GORD using proton pump inhibitors, H2-receptor antagonists or prokinetic agents. Participants had to be classifiable in the empirical treatment group (no endoscopy used in treatment allocation) or in the endoscopy negative reflux disease group (no endoscopic signs of erosive oesophagitis). DATA COLLECTION AND ANALYSIS: Data from included trials were extracted by two reviewers independently. The impact of interventions was expressed as relative risks (RR) together with 95% confidence intervals (95% CI). Meta-analysis was only performed if there were sufficient trials of similar comparisons reporting the same outcomes. Relative risks were combined for binary outcomes. MAIN RESULTS: Twenty-one trials were included: eleven in the empirical treatment group, seven in the ENRD group and three in both. In empirical treatment of GORD the RR for heartburn remission in placebo-controlled trials for PPI was 0.35 (1 trial, 95% CI 0.26 to 0.46), for H2RAs 0.77 (2 trials, 95% CI 0.60 to 0.99) and for prokinetics 0.86 (1 trial, 95% CI 0.73 to 1.01). In direct comparison PPIs were significantly (p<0.05) more effective than H2RAs (3 trials, RR 0.67, 95% CI 0.57 to 0.80) and prokinetic's (2 trials, RR 0.53, 95% CI 0.32 to 0.87). In treatment of ENRD, RR for heartburn remission for PPI versus placebo was 0.68 (4 trials, 95% CI 0.53 to 0.88) and for H2RA versus placebo was 0.84 (2 trials, 95% CI 0.74 to 0.95). The RR for PPI versus H2RA was 0.69 (2 trials, 95% CI 0.39 to 1.20) and versus prokinetic 0.72 (1 trial, 95% CI 0.56 to 0.92). REVIEWER'S CONCLUSIONS: The findings in this review suggest that antisecretory drugs are effective in the empirical treatment of complaints likely to originate from GORD and in treatment of ENRD and furthermore that PPIs are superior to H2RAs in empirical treatment of typical GORD symptoms.

Endoscopy, Digestive System↗
PubMed1987

Correlation of the turnover number of the ATP synthase in liposomes with the proton flux and the proton potential across the membrane.

The fluorescent indicator pyranine was used for recording the internal pH of liposomes. The proton permeability was deduced from the velocity of the internal pH increase which was caused by shifting the external pH from 7 to 9. From valinomycin titration of the proton permeability in the presence of internal and external KCl (0.1 M), the permeability coefficient of H+ (PH) was obtained as 10(-4) cm/s at 22 degrees C. The coefficient was twice this value with the ATP synthase isolated from Wolinella succinogenes present in the liposomal membrane (10 mg protein/g phospholipid). ADP and phosphate had no effect on the latter PH. The protonophore TTFB (5 mumol/g phospholipid) increased the PH by 3 orders of magnitude. The permeability coefficients of H+ and K+ were used for calculating the delta uH and the proton flux associated with the phosphorylation which was driven by gradients of H+ and K+. For the conditions of limiting permeability of K+, the following conclusions were drawn. (1) In the steady state of rapid ion flux, the electrical potential across the liposomal membrane as calculated according to the Goldman equation, is directed opposite to the corresponding Nernst potential which is calculated from the K+ gradient. (2) The maximum turnover numbers of phosphorylation require a delta uH of 200-220 mV across the liposomal membrane. These values of delta uH and the corresponding turnover numbers are close to those brought about by the bacterial electron transport and the coupled phosphorylation. (3) The velocity of phosphorylation is linearly related to the proton flux. The slope of the line can be explained on the basis of an H+/ATP ratio of approx. 3.

Arylsulfonates↗
PubMed1991

Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1.

We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits.

Biological Transport↗
PubMed1997

Proton pump activity of mitochondria-rich cells. The interpretation of external proton-concentration gradients.

We have hypothesized that a major role of the apical H(+)-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl- via an apical Cl-/HCO3(-)-exchanger. The activity of the H+ pump was studied by monitoring mucosal [H+]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 +/- 0.07 (mean +/- SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 +/- 18 microns, corresponding to an estimated "unstirred layer" thickness of 329 +/- 29 microns. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The [H+] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H+]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the [H+] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H+]-gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H+]-gradient depended on the mucosal anion such as to indicate that base (HCO3-) is excreted in exchange not only for Cl-, but also for Br- and I-, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.

Animals↗
PubMed2000

Proton affinity of peroxyacetyl nitrate. A computational study of topical proton affinities.

The structure and energetics of the peroxyacetyl nitrate conformers syn- and anti-PAN and several cations formed by PAN protonation were investigated by a combination of density functional theory and ab initio calculations. syn-PAN is the more stable conformer that is predicted to predominate in gas-phase equilibria. The acetyl carbonyl oxygen was found to be the most basic site in PAN, the oxygen atoms of the peroxide and NO(2) groups being less basic. The 298 K proton affinity of syn-PAN was calculated as 759-763 kJ mol(-1) by effective QCISD(T)/6-311 + G(3df,2p) and 771-773 kJ mol(-1) by B3-MP2/6-311 + G(3df,2p). The calculated values are 25-39 kJ mol(-1) lower than the previous estimate by Srinivasan et al. (Rapid Commun. Mass Spectrom. 1998; 12: 328) that was based on competitive dissociations of proton-bound dimers (the kinetic method). The calculated threshold dissociation energies predicted the formation of CH(3)CO(+) + syn - HOONO(2) and CH(3)COOOH + NO(2)(+) to be the most favorable fragmentations of protonated PAN that required 83 and 89 kJ mol(-1) at the respective thermochemical thresholds at 298 K. The previously observed dissociation to CH(3)COOH + NO(3)(+) was calculated by effective QCISD(T)/6-311 + G(3df,2p) to require 320 kJ mol(-1). The disagreement between the experimental data and calculated energetics is discussed.

Air Pollutants↗