scholarly journals Anion Conductance of Frog Muscle Membranes: One Channel, Two Kinds of pH Dependence

1973 ◽  
Vol 62 (3) ◽  
pp. 324-353 ◽  
Author(s):  
J. W. Woodbury ◽  
P. R. Miles
Keyword(s):  
Transmembrane Potential ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Complete Sequence ◽  
Minimum Size ◽  
Bathing Solution ◽  
Zinc Ions ◽  
Membrane Conductances ◽  
Anion Conductance ◽  
Characteristic Resistance

Anion conductance and permeability sequences were obtained for frog skeletal muscle membranes from the changes in characteristic resistance and transmembrane potential after the replacement of one anion by another in the bathing solution. Permeability and conductance sequences are the same. The conductance sequence at pH = 7.4 is Cl- Br- > NO3- > I- > trichloroacetate ≥ benzoate > valerate > butyrate > proprionate > formate > acetate ≥ lactate > benzenesulfonate ≥ isethionate > methylsulfonate > glutamate ≥ cysteate. The anions are divided into two classes: (a) Chloride-like anions (Cl- through trichloroacetate) have membrane conductances that decrease as pH decreases. The last six members of the complete sequence are also chloride like. (b) Benzoate-like anions (benzoate through acetate) have conductances that increase as pH decreases. At pH = 6.7 zinc ions block Cl- and benzoate conductances with inhibitory dissociation constants of 0.12 and 0.16 mM, respectively. Chloride-like and benzoate-like anions probably use the same channels. The minimum size of the channel aperture is estimated as 5.5 x 6.5 Å from the dimensions of the largest permeating anions. A simple model of the channel qualitatively explains chloride-like and benzoate-like conductance sequences and their dependence on pH.

scholarly journals THE pH DEPENDENCE AND DISSOCIATION CONSTANTS OF ESTERASES AND PROTEASES TREATED WITH DIISOPROPYL FLUOROPHOSPHATE

1957 ◽  
Vol 226 (2) ◽  
pp. 867-872 ◽  
Author(s):  
L.A. Mounter ◽  
H.C. Alexander ◽  
Kenneth D. Tuck ◽  
Lien Tien H. Dien
Keyword(s):  
Dissociation Constants ◽  
Ph Dependence ◽  
Diisopropyl Fluorophosphate

scholarly journals Mannitol-1-phosphate dehydrogenase of Escherichia coli Chemical properties and binding of substrates

1986 ◽  
Vol 239 (2) ◽  
pp. 435-443 ◽  
Author(s):  
T Chase
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Covalent Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thiol Group ◽  
Covalent Modification ◽  
Protective Effects ◽  
Phosphate Complex

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5′-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5′-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.

scholarly journals Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A

1993 ◽  
Vol 289 (1) ◽  
pp. 185-193 ◽  
Author(s):  
W L Mock ◽  
D J Freeman ◽  
M Aksamawati
Keyword(s):  
Metal Ion ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Bound Water ◽  
Acid Dissociation ◽  
Lewis Acidity ◽  
Carboxypeptidase A ◽  
A Value ◽  
Straight Line

Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.

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.

Myotonia and block of chloride conductance by iodide in avian muscle

1975 ◽  
Vol 229 (5) ◽  
pp. 1155-1158 ◽  
Author(s):  
KG Morgan ◽  
RK Entrikin ◽  
SH Bryant
Keyword(s):  
Skeletal Muscle ◽  
Drinking Water ◽  
Mechanical Stimulation ◽  
Muscle Fibers ◽  
Potassium Conductance ◽  
Repetitive Firing ◽  
Membrane Conductances ◽  
Anion Conductance ◽  
Avian Muscle

Immature chickens and adult pigeons whose drinking water contained 3% KI for 1--10 days developed myotonia, characterized by stiffness on sudden movement and abnormal repetitive firing of skeletal muscle fibers. Component resting membrane conductances, excitability, and membrane potentials of biventer cervicis muscle fibers from adult pigeons were measured in vitro at 38--39degreesC. Fibers from iodide-treated pigeons in normal solution and fibers from untreated pigeons in I--containing solution (15--120 mM) responded repetitively to electrical and mechanical stimulation. Resting anion conductance (Ganion), assumed to be the sum of C1- and I- conductances, of fibers from iodide-treated pigeons decreased nonlinearly from 2,565 to 266 mumho/cm2 when the bath concentration of I- was increased from 0 to 120 mM. Potassium conductance was assumed constant at 577 mumho/cm2. Ganion of fibers from iodide-treated pigeons was 50% of control and equaled that of untreated fibers in 15 mM I- containing medium. Reduction of the stabilizing Ganion and increased mechanical responsiveness can account for the iodide-induced myotonia in birds.

pH sensitivity of the basolateral membrane of the rabbit proximal tubule

1986 ◽  
Vol 250 (2) ◽  
pp. F261-F266 ◽  
Author(s):  
B. A. Biagi ◽  
M. Sohtell
Keyword(s):  
Potassium Concentration ◽  
Ph Dependence ◽  
Basolateral Membrane ◽  
Rabbit Kidney ◽  
Bathing Solution ◽  
Solution Ph ◽  
Absolute Value ◽  
Ph Dependent ◽  
Basolateral Membrane Potential

Conventional microelectrodes were used to study the effects of bath pH and bicarbonate concentrations on the basolateral membrane potential (Vbl) of cells from the superficial proximal convoluted (PCT) and proximal straight (PST) tubules of the rabbit kidney perfused in vitro. Bathing solution pH was varied over the range of 5.9-7.4 using either control (22-25 mM) or low bicarbonate (5.0-6.6 mM) Ringer solutions and the appropriate CO2 tensions. The results show a strong pH dependence of the steady-state values of Vbl in both the convoluted and straight tubule segments. The pH-dependent depolarization was approximately 35 mV/pH unit change of the bathing solution in the acid direction and could be demonstrated in CO2-free HEPES-buffered solutions. A depolarizing response to increased bath potassium concentration (HK) was observed that was linearly related to the absolute value of the Vbl under control conditions. Under acidotic conditions, reduced HK depolarizations indicate that a decrease in the relative potassium permeability of the basolateral membrane is the principle mechanism underlying the effects of bath pH on Vbl.

Electrophysiological and optical changes in slices of rat hippocampus during spreading depression

1983 ◽  
Vol 50 (3) ◽  
pp. 561-572 ◽  
Author(s):  
R. W. Snow ◽  
C. P. Taylor ◽  
F. E. Dudek
Keyword(s):  
Time Course ◽  
Transmembrane Potential ◽  
Spreading Depression ◽  
Pyramidal Neurons ◽  
Input Resistance ◽  
Rat Hippocampus ◽  
Bathing Solution ◽  
Normal Medium ◽  
Repetitive Electrical Stimulation ◽  
Similar Decrease

Spreading depression (SD) was studied with intracellular and extracellular recordings and with photometry in slices of rat hippocampus. Repetitive electrical stimulation could initiate SD in either normal medium or in low-Ca2+ medium containing Mn2+, especially during transient hypoxia. The extracellular voltage near CA1 pyramidal somata and dendrites became negative by approximately 18 mV during SD. This negativity peaked more slowly in low-Ca2+ medium containing Mn2+. The wave of negativity propagated across the slice in both media at approximately 6 mm/min. Input resistance of pyramidal neurons became undetectable during SD, and differential voltage recording between neurons and adjacent extracellular space demonstrated that transmembrane potential approached zero. Slices became more opaque during SD. Photometry revealed approximately 10% increase in reflectance and a similar decrease in transmittance of white light, which occurred with a time course similar to the extracellularly recorded voltage shift. These data support the hypothesis that SD represents a large increase in membrane permeability associated with substantial movements of water. The persistance of SD in a bathing solution that blocked electrically evoked postsynaptic potentials suggests that the contribution of synaptic transmitter release to the propagation of SD should be reappraised.

Symmetric pH dependence of buffering power in giant fused cells from frog kidney proximal tubule

1997 ◽  
Vol 272 (4) ◽  
pp. R1226-R1234 ◽  
Author(s):  
P. Bouyer ◽  
M. Cougnon ◽  
S. R. Thomas ◽  
P. Hulin ◽  
T. Anagnostopoulos ◽  
...  
Keyword(s):  
Transmembrane Potential ◽  
Ph Dependence ◽  
Cell Type ◽  
Frog Kidney ◽  
Apparent Dissociation Constant ◽  
Buffering Power ◽  
Alkaline Cell ◽  
A Cell ◽  
Proximal Kidney Tubule ◽  
Transmembrane Potential Difference

This study measures the intrinsic buffering power (beta(i)) of giant fused cells from the proximal kidney tubule of the frog (Rana ridibunda) as a function of intracellular pH (pHi). We monitored pHi and transmembrane potential difference during acid or alkaline cell loading, achieved by removal of NH4Cl-containing solutions or CO2-HCO3(-)-equilibrated solutions, respectively, in the absence of extracellular Na+. Data were well fit by the equation for a single, monoprotic buffer with a maximum beta(i) at a pHi of 7.39 +/- 0.06 and a total buffer concentration of 30.7 +/- 1.6 mM (means +/- SD). From pHi measurements obtained during CO2-HCO3- exposure, we also calculated the buffering power afforded by the CO2-HCO3- pair, and we show its increasing contribution to total buffering power at increasing PCO2 and pHi. To our knowledge, this is the first report of a cell type in which intrinsic cell buffers can be adequately approximated as a single monoprotic buffer with a negative logarithm of apparent dissociation constant in the normal physiological range and essentially symmetric dependence on pHi in both acid and alkaline ranges.

scholarly journals Characterization of retinaldehyde dehydrogenase 3

2006 ◽  
Vol 394 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Caroline E. Graham ◽  
Keith Brocklehurst ◽  
Richard W. Pickersgill ◽  
Martin J. Warren
Keyword(s):  
Carboxy Group ◽  
Dissociation Constants ◽  
Homology Modelling ◽  
Ph Dependence ◽  
Tryptophan Fluorescence ◽  
Aldehyde Group ◽  
Base Catalysis ◽  
Ligand Docking ◽  
Binding Studies ◽  
Macroscopic Pka

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488.

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