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1.
John Gutknecht 《Journal of bioenergetics and biomembranes》1987,19(5):427-442
The proton/hydroxide (H+/OH–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H+/OH– transport are unknown. This review describes the characteristics of H+/OH– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H+/OH– conductances (G
H/OH) range from 2–6 nS cm–2, corresponding to net H+/OH– permeabilities of (0.4–1.7)×10–5 cm sec–1. Inhibitors ofG
H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG
H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG
H/OH are not correlated. The characteristics ofG
H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG
H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG
H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG
H/OH remains which may be due to water wires, hydrated defects, or other mechanisms. 相似文献
2.
John Gutknecht 《The Journal of membrane biology》1984,82(1):105-112
Summary A simple method of measuring proton/hydroxide conductance (G
H/OH) through planar lipid bilayer membranes is described. First the total conductance (G
m
) is measured electrically. Then the H+/OH– transference number (T
H/OH) is estimated from the diffusion potential (V
m
) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H+ and OH–. Thus,V
m
is due entirely to H+/OH– diffusion andG
H/OH can be calculated from the relations,V
m
=T
H/OH
E
H/OH andG
H/OH=T
H/OH
G
m
, whereE
H/OH is the equilibrium potential for H+ and OH–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G
H/OH is nearly independent of pH, ranging from about 10–9 S cm–2 at pH 1.6 to 10–8 S cm–2 at pH 10.5. BecauseG
H/OH is nearly independent of pH, the calculated permeability coefficients to H+ and/or OH– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G
H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG
H/OH.G
H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H+/OH– conductance is not known, the relation betweenG
H/OH and water activity suggests that several water molecules are involved in the H+/OH– transport process. 相似文献
3.
Summary By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 F 1 cm2 actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 F for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (I
sc) in these tight epithelia.G andI
sc were increased by mucosal (Na+) [I
sc0 when (Na+)0], aldosterone, serosal (HCO
3
–
) and high mucosal (H+); were decreased by amiloride, mucosal (Ca++), ouabain, metabolic inhibitors and serosal (H+); and were unaffected by (Cl–) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na+ intake caused variations in bladderG andI
sc similar to those caused by the expectedin vivo changes in aldosterone levels. The relation betweenG andI
sc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from thisG–I
sc relation. Net Na+ flux equalledI
sc. Net Cl– flux was zero on short circuit and equalled only 25% of net Na+ flux in open circuit. Bladder membrane fragments contained a Na+–K+-activated, ouabain-inhibited ATPase. The physiological significance of Na+ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na+-depleted. 相似文献
4.
F. Thévenod T. P. Kemmer A. L. Christian I. Schulz 《The Journal of membrane biology》1989,107(3):263-275
Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K
m
for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulatioin was abolished by protonophores. such as nigericin (10–6 mol/liter) or CCCP (10–5 mol/liter), and by inhibitors of nonmitochondria H+ ATPase, such as NEM or NBD-Cl, at a concentration of 10–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2×10–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10–4 mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependernt in the sequence Cl–>Br–>gluconate–; in the presence of SO
4
–2
. CH3COO– or No
3
–
, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+>Li+>Na+=choline+, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl– and a K+ conductance, as well as a small H+ conductance. were found in the microsomal membranes. When membranes containing the H+ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H+ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes. 相似文献
5.
Paddock M.L. Senft M.E. Graige M.S. Rongey S.H. Turanchik T. Feher G. Okamura M.Y 《Photosynthesis research》1998,55(2-3):281-291
The structural basis for proton coupled electron transfer to QB in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k
AB
(2)(QA
–QB
– + H+ QAQBH–) by at least 103 (pH 7.5) and a recombination rate k
BD (D+QAQB
– DQAQB) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k
AB
(2) on the free energy for electron transfer (Get). k
AB
(2) was independent of Get in DN(L213) RCs, but dependent on Get in native and all suppressor RCs. This shows that proton transfer limits the k
AB
(2) reaction with a rate of 0.1s–1 in DN(L213) RCs but is not rate limiting and at least 108-fold faster in native and 105-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near QB
–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near QB. 相似文献
6.
Summary We demonstrate that a simple kinetic model describes the transport of protons across lipid bilayer membranes by the weak acid CCCP (carbonyl cyanidem-chlorophenylhydrazone). Four parameters characterize this model: the adsorption coefficients of the anionic and neutral forms of the weak acid onto the interface (
A and
HA) and the rate constants for the movement of A– and HA across the membrane (k
A andk
HA). These parameters were determined by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. From these equilibrium and steady state measurements on diphytanoyl phosphatidylcholine/chlorodecane membranes we found that
A=
HA=1.4 10-3cm,k
A=175 s–1 andk
HA=12,000 sec–1. These parameters and our model describe our kinetic experiments if we assume that the protonation reactions, which occur at the interfaces, remain at equilibrium. The model predicts a single exponential decay of the current in a voltage-clamp experimetn. The model also predicts that the decay in the voltage across the membrane following an intense current pulse of short duration (50 nsec) can be described by the sum of two exponentials. The magnitudes and time constants of the relaxations that we observed in both voltage-clamp and charge-pulse experiments agree well with the predictions of the model for all values of pH, voltage and [CCCP]. 相似文献
7.
Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na+K+-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg2+-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na+K+-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na+K+-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca2+-ATPase of native erythrocyte membranes (i.e., increase ofV
max from 1.6 to 5 mol PO
4
3–
·phospholipid–1·hr–1, decrease of K
Ca
from 6 m to 1.4–1.8 m). Stearic acid decreasedK
Ca
(2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg2+-ATPase, Na+K+-ATPase, and Ca2+-ATPase could be achieved by incubation of the membranes with phospholipase A2.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results. 相似文献
8.
At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho
+ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho
– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho
+ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho
– cells in the population is very high. This does not result in an increase in the frequency ofrho
– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho
– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho
+ andrho
– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho
– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho
– mutants are less viable thanrho
+ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho
– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol. 相似文献
9.
David H. Thomas Erik Skadhauge 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1989,159(1):51-60
Summary The function of the caecal bulb, and its adaptation to chronic high- or low-Na+ intake, was investigated by in vivo perfusion of anaesthetised birds. Effects of acute aldosterone injection (125 g·kg–1 body mass) were also measured.Evidence was found for primary active net absorption of Na+, inducing parallel Na-linked absorption of water and Cl– and secretion of K+. Around 20–35% of total Cl– absorption and K+ secretion were independent of Na+ fluxes, and these components appear to be driven by passive processes with apparent conductances of 6.3×10–3 (G
Cl) and 1.1×10–3 (G
K) S·cm–2.Acetate (40mM) stimulated Na+ fluxes (8.5–9.9 Eq·cm–2·h–1) and Na-linked water fluxes (27–44 l·cm–2·h–1). Increased coupling ratios (2.9–4.6 l·Eq–1) and other data indicate that these effects may be due to increased osmotic permeabilities of barriers involved in the Na-linked water transfer pathway.Low-Na+ maintenance enhanced EPD (49–69 mV, serosa positive) and all net fluxes:J
Na (6.8–11.6);J
K (–3.2––4.3);J
Cl (4.3–5.6 Eq·cm serosal area–2·h–1);J
v (28–43 l·cm–2·h–1) (mucosal-serosal fluxes positive).Acute aldosterone enhancedJ
Na (10.8–14.0 Eq·cm–2·h–1) and EPD (54–66 mV) by 3 h after injection, but had no effect on the Na-linked components ofJ
K orJ
Cl.Abbreviations
ECPD, EPD
Electrochemical or electrical potential difference
-
G
Cl
,G
K
ionic conductances (Cl–, K+)
-
J
v
,J
ion
net volume or ion flux rate, mucosa-serosa positive;P
d
(Cl) diffusive permeability coefficient (of Cl–)
-
SEDM
standard error of difference between means 相似文献
10.
Gotthold Gäbel Susanne Vogler Holger Martens 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(4):419-426
Summary Unidirectional 22Na+ and 36Cl– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO2/HCO
–
3
-containing and CO2/HCO
–
3
-free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na+ absorption without affecting the Cl– transport in the same direction. Short-chain fatty acid stimulation of Na+ transport was at least partly independent of Cl– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na+ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO2 in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na+ flux. Along with its effect on sodium transport, raising the PCO2 also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO2 permeate the cell membrane and produce a raise in intracellular H+ concentration. This stimulates an apical Na+/H+ exchange, leading to increased Na+ transport. The stimulatory effect of CO2 on Cl– transport is probably mediated by a Cl–/HCO
–
3
exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations
DIDS
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
-
G
t
transepithelial conductance (mS·cm-2)
-
HSCFA
undissociated short-chain fatty acids
-
J
ms
mucosal-to-serosal flux (Eq · cm-2 · h-1)
-
J
net
net flux (Eq · cm-2 · h-1)
-
J
sm
serosal-to-mucosal flux (Eq · cm-2 · h-1)
-
PD
transepithelial potential difference (mV)
-
SCFA
dissociated short-chain fatty acids
-
SCFA
short-chain fatty acids 相似文献
11.
Garnett Trevor P. Shabala Sergey N. Smethurst Philip J. Newman Ian A. 《Plant and Soil》2001,236(1):55-62
Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH4
+, NO3
– and H+ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 m NH4NO3. The pattern of fluxes was such that there was a large influx of NH4
+, a smaller influx of NO3
– and large H+ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:–6 (NH4
+:NO3
–:H+). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH4
+ and H+, and NH4
+ and NO3
–. Overall, these results are consistent with NH4
+ being the preferred source N for E. nitens. 相似文献
12.
Aspirin,acetaminophen and proton transport through phospholipid bilayers and mitochondrial membranes
John Gutknecht 《Molecular and cellular biochemistry》1992,114(1-2):3-8
Mechanisms of proton transport were investigated in planar phospholipid bilayer membranes exposed to aspirin (acetylsalicylic acid), acetaminophen (4-acetamidophenol), benzoic acid and three aspirin metabolites (salicylic acid, gentisic acid and salicyluric acid). The objectives were to characterize the conductances and permeabilities of these weak acids in lipid bilayer membranes and then predict their effects on mitochondrial membranes. Of the compounds tested only aspirin, benzoate and salicylate caused significant increases in membrane conductance. The conductance was due mainly to proton current at low pH and to weak acid anion current at neutral pH. Analysis of the concentration and pH dependence suggests that these weak acids act as HA2
–-type proton carriers when pH pK and as lipid soluble anions at neutral pH. Salicylate is much more potent than aspirin and benzoate because salicylate contains an internal hydrogen bond which delocalizes the negative charge and increases the permeability of the anion. Model calculations for mitochondria suggest that salicylate causes net H+ uptake by a cyclic process of HA influx and A– efflux. This model can explain the salicylate-induced uncoupling and swelling observed in isolated mitochondria. Since ingested aspirin breaks down rapidly to form salicylate, these results may clarify the mechanisms of aspirin toxicity in humans. The results may also help to explain why the ingestion of aspirin but not acetaminophen is associated with Reye's syndrome, a disease characterized by impaired energy metabolism and mitochondrial swelling. 相似文献
13.
Michael R. Blatt Alonso Rodriguez-Navarro Clifford L. Slayman 《The Journal of membrane biology》1987,98(2):169-189
Summary Active transport of potassium in K+-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K+ pumps or K+/H+-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H+ to 1 K+ taken inward so that K+, but not H+, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol.
87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K+–H+ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K+ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm2) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K+-H+ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K+ transport and K+-H+ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K+-H+ currents. Thus, the inhibition of K+ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K+]o and pHo on theI–V curve for K+-H+ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K+, butincreases affinity for protons (K
m
range: for [K+]o, 15–45 M; for [H+]o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K+ first and H+ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H+ first and K+ last. 相似文献
14.
Summary The area-specific coductance of the membrane in the acid and basic zones (denoted byG
A
andG
B
, respectively) ofChara cells was measured in flowing solutions, containing 5mm zwitterionic buffer, as a function of the external pH(denoted by pH0). During illuminationG
A
was 1 S/m2 for pH0 in the range 5 to 8.5, and increased markedly to 3 to 6 S/m2 at higher pH0.G
B
, however, was always larger thanG
A
during illumination with a typical magnitude of 5 to 15 S/m2 for pH0 6 to 12. Thus under many experimental conditions it is possible that there is no single correct value for the membrane area-specific conductance. A flow of current in the external medium between the acid and basic regions was found to be associated with pH banding, and also withG
B
exceedingG
A
. This current could be present in flowing solutions without added HCO
3
–
over a wide range of pH0 and at high (25mm) buffer concentration. Combining measurements ofG
A
andG
B
with measurements of the currents in the acid and basic zones (denoted byJ
A
andJ
B
, respectively), it was estimated that the resting (i.e. in the absence of net current flow) potential difference (PD) across the membranes within the individual zones (denoted byU
A
andU
B
) was –265±20 and –183±5 mV, respectively, during illumination. Upon the removal of illumination at pH0-7.5,G
A
,G
B
andJ
B
were found to decrease rapidly during the initial few hundred seconds. During this period (U
B
–V
m
) remained relatively constant. A transient hyperpolarization ofV
m
often occurred, the magnitude of which was correlated with the magnitude ofJ
B
prior to the removal of illumination. After some 0.5 to 1 ksec of darkness,G
A
andG
B
had both decreased considerably and nowG
A
G
B
andU
A
U
B
V
m
. Eventually, after 2 to 8 ksec of darkness, the membrane conductance was effectively homogeneous with a much smaller magnitude (typically<0.2S/m2) andV
m
was depolarized by typically 5 to 15 mV. 相似文献
15.
Summary Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes.We measured transepithelial impedance under three conditions: (1) control conditions in which the epithelium was opencircuited and bathed on both sides with NaCl–HCO3 Ringer's solutions, (2) amiloride conditions which were similar to control except that 100 m amiloride was present in the mucosal bathing solution, and (3) mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate (K+ Ringer's). Three morphologically-based equivalent circuit models were used to evaluate the data: (1) a lumped model (which ignores LIS resistance), (2) a LIS distributed model (distributed basolateral membrane impedance) and (3) a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (G
s
) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12–15% underestimate of membrane resistance ratio (R
a
/R
b
) for the control and amiloride conditions and a 34% underestimate for the K Ringer's condition. Distributed resistance effects arising from the crypts would produce a 1–2% overestimate ofR
a
/R
b
.Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 F/cm2 and the mean apical membrane specific conductance (G
a-norm) was 0.17 mS/F. The average basolateral membrane capacitance was 11 F/cm2 with a mean specific conductance (G
b-norm) of 1.27 mS/F.G
a-norm was decreased by amiloride or K+ ringer's to 0.07 mS/gmF and 0.06 mS/F, respectively. Basolateral conductance was also reduced by amiloride, whereas capacitance was unchanged (G
b-norm=0.97 mS/F). For the K+ Ringer's condition, both basolateral conductance and capacitance were greatly increased such thatG
b-norm was not significantly different from the control condition. 相似文献
16.
The energetics of the first stable charge separated state, P+QA– relative to that of P–QA was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–QA and P+QA– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P+QA– state runs parallel to the (integrated) net proton uptake due to the PQA/P+QA– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pKa values as well as the light-induced pKa changes of the protonatable groups of the protein. The observed dependence of the energetics of P+QA– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt. 相似文献
17.
The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H+/e
– stoichiometry of the pump is discussed and results [Wikström (1989),Nature
338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. Direct and indirect mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the CuA site. 相似文献
18.
Monocarboxylic acid permeation through lipid bilayer membranes 总被引:1,自引:0,他引:1
Summary The membrane permeability coefficients for the homologous monocarboxylic acids, formic through hexanoic, as well as benzoic and salicylic, were determined for egg phosphatidylcholine-decane planar bilayer membranes. The permeabilities of formic, acetic and propionic acid were also determined for solvent-free phosphatidylethanolamine bilayers. Permeability coefficients were calculated from tracer fluxes measured under otherwise symmetrical conditions, and precautions were taken to ensure that the values were not underestimated due to unstirred layer effects. The relation between the nonionic (HA) permeability (P
m
) and the hexadecane/water partition coefficient (K
p
) was: log
m
=0.90 log Kp+0.87 (correlation coefficient=0.996). Formic acid was excluded from the analysis because its permeability was sixfold higher than predicted by the other acids. The permeabilities for solvent-free membranes were similar to those for decanecontaining membranes. The exceptionally high permeability of formic acid and the high correlation of the other permeabilities to the hexadecane/water partition coefficient is a pattern that conforms with other nonelectrolyte permeabilities through bilayers. Similarly, the mean incremental free energy change per methylene group (G-CH2-) was –764 cal mol–1, similar to other homologous solutes in other membrane systems. However, much less negative G values (–120, to –400 cal mol–1) were previously reported for fatty acids permeating bilayers and biological membranes. These values are due primarily to unstirred layer effects, metabolism and binding to membranes and other cell components. 相似文献
19.
The primary act of charge separation was studied in P+BA
– and P+HA
– states (P, primary electron donor; BA and HA, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of BA
– (1020 nm) and of absorption band of HA (760 nm). It was found that in native RCs kept in heavy water (D2O) buffer the isotopic decreasing of basic oscillation frequency 32 cm –1 and its overtones takes place by the same factor 1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H2O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm –1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor PB and primary electron acceptor BA. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to BA. The results allow for tracing of the possible pathway of electron transfer from P* to BA along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(PB)-N-C-N(His M200)-HOH-O = BA. We assume that the role of 32-cm –1 modulation in electron transfer along this chain consists of a fixation of electron density at BA
– during a reversible electron transfer, when populations of P* and P+BA
– states are approximately equal. 相似文献
20.
Gopta Oksana A. Cherepanov Dmitry A. Mulkidjanian Armen Y. Semenov Alexey Yu. Bloch Dmitry A. 《Photosynthesis research》1998,55(2-3):309-316
Direct electrometry was used to study the light-induced voltage changes in the Rhodobacter sphaeroides chromatophores adsorbed to a phospholipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was followed by a slower increase attributed to the proton uptake in the QB site of the photosynthetic reaction centers. Kinetics and relative amplitudes of these voltage changes attributed to the QA
–.
B
–. QAQBH2 transition, were measured as a function of pH and temperature between +4 and +40 °C. The kinetics can be approximated by a single exponent above +23 °C (100 µs at +25 °C, pH 7.2), whereas below this temperature, it was a good fit of two exponential approximation (65 µs and 360 µs with similar contributions at +10 °C, pH 7.2). The faster component diminished with an apparent pK 8.5, whereas the slower one was maintained at a constant level until pH 9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower component (55 – 65 kJ/mol) was much higher than that of the faster component (< 10 kJ/mol). The two voltage components can be attributed to the transfer of the first (faster component) and the second (slower component) proton from the reaction center surface to QB. We suggested that higher activation energy of the slower component was due to a conformational change in the reaction center kinetically coupled to the second proton transfer to QBH–.The faster component diminished in the presence of 1 M KCl, with an apparent pK 7.5. To explain this observation, we assume that: (i) the midpoint potential of the QA/QA
–. redox pair was higher in 1 M KCl because of the reduced surface potential of chromatophores; (ii) the midpoint potential of the QB
–./QBH–. redox pair was insensitive to the surface potential change; (iii) the equilibrium constant of the reaction QA
–.QB
–. QAQBH– decreased at high ionic strength. 相似文献