scholarly journals Deuterium Isotope Effects on Permeation and Gating of Proton Channels in Rat Alveolar Epithelium

1997 ◽  
Vol 109 (4) ◽  
pp. 415-434 ◽  
Author(s):  
Thomas E. DeCoursey ◽  
Vladimir V. Cherny
Keyword(s):  
Deuterium Oxide ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Channel Gating ◽  
Alveolar Epithelium ◽  
Ionic Species ◽  
Alveolar Epithelial Cells ◽  
Proton Channels ◽  
Deuterium Isotope Effects ◽  
Solvent Isotope

The voltage-activated H+ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D2O, for water, H2O, on both the conductance and the pH dependence of gating were explored. D+ was able to permeate proton channels, but with a conductance only about 50% that of H+. The conductance in D2O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D+ than H+), suggesting that D+ interacts specifically with the channel during permeation. Evidently the H+ or D+ current is not diffusion limited, and the H+ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H+ (or D+) and not OH− is the ionic species carrying current. The voltage dependence of H+ channel gating characteristically is sensitive to pHo and pHi and was regulated by pDo and pDi in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H+ current activation was about three times slower (τact was larger) in D2O than in H2O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H+ channel activation requires deprotonation of the channel. In contrast, deactivation (τtail) was slowed only by a factor ≤1.5 in D2O. The results are interpreted within the context of a model for the regulation of H+ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D2O can be explained if the pKa of the external regulatory site is ∼0.5 pH U higher in D2O.

Mechanism of initiation in the thermal polymerization of styrene. Kinetic deuterium isotope effects in the initiation step of the thermal polymerization of some deuterated styrenes

1969 ◽  
Vol 47 (21) ◽  
pp. 4049-4058 ◽  
Author(s):  
Karl R. Kopecky ◽  
Syamalarao Evani
Keyword(s):  
Hydrogen Transfer ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Thermal Polymerization ◽  
Convenient Synthesis ◽  
Initiation Step ◽  
Deuterium Isotope Effects

A convenient synthesis of 2,6-dideuteriostyrene starts with N,N-dimethyl-(1-phenylethyl)-amine which is deuterated in the 2 and 6 positions by a series of exchanges using n-butyllithium followed by deuterium oxide. The deuterium isotope effects at 70° on the rates of the thermal polymerization, [Formula: see text], of 2,6-dideuterio-, α-deuterio-, and β,β-dideuteriostyrene are 1.29, 1.00, and 0.78, respectively. The deuterium isotope effects at 70° on the 2,2′-azobis-(2-methylpropionitrile) initiated rates of polymerization,[Formula: see text], are 0.96, 0.86, and 0.81, respectively. From these values the deuterium isotope effects on the rates of initiation of the thermal polymerization, k1H/k1D, are calculated to be 1.80, 1.31, and 0.92, respectively. At 147° the presence of 1.5% potassium t-butoxide decreases the rate of the thermal polymerization of neat styrene by a factor of 17, and results in the formation of 1-phenyltetralin as the greatly predominant dimer. The results support the suggestion that the thermal polymerization of styrene is initiated by hydrogen transfer from 1-phenyl-1,2,3,9-tetrahydronaphthalene, formed by a concerted dimerization of two molecules of styrene, to a third molecule of styrene.

Voltage-gated proton channels help regulate pHi in rat alveolar epithelium

2005 ◽  
Vol 288 (2) ◽  
pp. L398-L408 ◽  
Author(s):  
Ricardo Murphy ◽  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Thomas E. DeCoursey
Keyword(s):  
Epithelial Cells ◽  
Ph Regulation ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Proton Channel ◽  
Resting Cells ◽  
Proton Channels ◽  
Voltage Gated ◽  
Alveolar Epithelial

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pHi) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO3−-free solutions [at external pH (pHo) 7.4]. Cells were acid-loaded by the NH4+ prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 μM ZnCl2, a proton channel inhibitor, slowed recovery about twofold. In addition, the pHi minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl2 was observed at pHo 7.4 and pHo 8.0 and in normal and high-K+ Ringer solutions. The observed rate of Zn2+-sensitive pHi recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pHi recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl2 had no effect on pHi in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pHi recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

The Hydrolysis of Disubstituted Alkyl Phosphorochloridates and N,N,N′,N′-Tetra-stibstituted Phosphorodiamidic Chlorides in Water and Deuterium Oxide: Heat Capacity of Activation and Kinetic Solvent Isotope Effects

1973 ◽  
Vol 51 (4) ◽  
pp. 597-603 ◽  
Author(s):  
E. C. F. Ko ◽  
R. E. Robertson
Keyword(s):  
Heat Capacity ◽  
Thermodynamic Parameters ◽  
Alkyl Group ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Potential Relationship ◽  
Mechanism Of Reaction ◽  
Solvent Isotope ◽  
Hydrolysis Of ◽  
Solvent Isotope Effects

The pseudo-thermodynamic parameters, ΔH≠, ΔS≠, and ΔCp≠ and the kinetic solvent isotope effects have been determined for the three alkyl-phosphorochloridates, where the alkyl group is ethylisopropyl and n-propyl; for tetra-methyl and tetra-ethyl phosphorodiamidic chlorides; the di-n-propyl and di-isopropyl analog, the di(isopropylmethylcarbinyl)phosphorochloridate and the tetra-ethylthiophosphorodiamidic chloride. These compounds have a potential relationship to compounds used as insecticides and as polymers. The mechanism of reaction is discussed on the basis of these data.

Solvent isotope effects in the oxidation of dipeptides by aqueous chlorine

1999 ◽  
Vol 77 (5-6) ◽  
pp. 997-1004 ◽  
Author(s):  
X L Armesto ◽  
M Canle L. ◽  
V García ◽  
J A Santaballa
Keyword(s):  
Isotope Effect ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Second Step ◽  
Solvent Isotope Effect ◽  
General Base ◽  
Peptide Oxidation ◽  
Solvent Isotope ◽  
Hydrolysis Of ◽  
Solvent Isotope Effects

A kinetic study of the mechanism of oxidation of Ala-Gly and Pro-Gly by aqueous chlorine has been carried out. Among other experimental facts, the deuterium solvent isotope effects were used to clarify the mechanisms involved. In a first stage, N-chlorination takes place, and then the (N-Cl)-dipeptide decomposes through two possible mechanisms, depending on the acidity of the medium. The initial chlorination step shows a small isotope effect. In alkaline medium, two consecutive processes take place: first, the general base-catalyzed formation of an azomethine (β ca. 0.27), which has an inverse deuterium solvent isotope effect (kOH-/kOD- ~ 0.8). In a second step, the hydrolysis of the azomethine intermediate takes place, which is also general base-catalyzed, without deuterium solvent isotope effect, the corresponding uncatalyzed process having a normal deuterium solvent isotope effect (kH2O/kD2O ~ 2). In acid medium, the (N-Cl)-dipeptide undergoes disproportionation to a (N,N)-di-Cl-dipeptide, the very fast decomposition of the latter in deuterium oxide preventing a reliable estimation of the solvent isotope effect.Key words: chlorination, deuterium isotope effects, fractionation factors, peptide oxidation, water treatment.

Deuterium Oxide Solvent Isotope Effects on Fast Reactions of Substituted Malonic Acids1

1966 ◽  
Vol 70 (11) ◽  
pp. 3490-3493 ◽  
Author(s):  
Melvin H. Miles ◽  
Edward M. Eyring ◽  
William W. Epstein ◽  
Michael T. Anderson
Keyword(s):  
Deuterium Oxide ◽  
Isotope Effects ◽  
Fast Reactions ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

Horseradish Peroxidase. XVII. Reactions of Compounds I and II with p-Aminobenzoic Acid in Deuterium Oxide

1975 ◽  
Vol 53 (11) ◽  
pp. 1563-1569 ◽  
Author(s):  
C. D. Hubbard ◽  
H. B. Dunford ◽  
W. D. Hewson
Keyword(s):  
Horseradish Peroxidase ◽  
Dissociation Constants ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Enzyme Concentration ◽  
Acid Dissociation ◽  
Aminobenzoic Acid ◽  
Kinetic Isotope

The kinetics of reactions of horseradish peroxidase compounds I and II (HRP-I and HRP-II, respectively) with p-aminobenzoic acid have been studied in ordinary water and in deuterium oxide solution over a pH (pD) range 3–10, at 25° and at an ionic strength of 0.11. Under the conditions of the experiments the rate of reaction is first order both in substrate concentration and in enzyme concentration in both solvents. An analysis of the pH dependence of the second order rate constant in H2O confirms the presence of two acid dissociation groups on the enzyme with pKa' of 8.6 and ∼0 for HRP-II, whereas for HRP-I the data suggest a pKa of 5.1 on the enzyme and reveal, as previously shown, the influence of the ionization of the substituted ammonium group of the substrate. In deuterium oxide the pD profiles are similar to those in water but significant shifts for both kinetic and acid dissociation constants are observed for both compounds.The numerical values of the isotope effects taken together with previous results in general confirm that with labile substrates the group of pKa 8.6 in H2O on HRP-II is involved in general acid catalysis. p-Aminobenzoic acid is intermediate between labile and somewhat unreactive substrates and behaves similarly to the ferrocyanide ion in that both acid dissociation groups (pKa's 8.6 and ∼0) are influential in the catalysis of substrate oxidation by HRP-II. The kinetic isotope effect for the HRP-I reaction with p-aminobenzoic acid at high pH (pD) is consistent with a rate determining proton transfer but the group of pKa 5.1 in H2O remains unidentified.

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