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1.
Summary Microplasmodia ofPhysarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30mm K+, 4mm Ca++, 3mm Mg++, 18mm citrate buffer, pH 4.7, 22°C), the transmembrane potential differenceV
m is around –100 mV and the membrane resistance about 0.25 m2.V
m is insensitive to light and changes of the Na+/K+ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN–, N
3
–
),V
m drops to about 0 mV. Under normal conditions,V
m is very sensitive to external pH (pH
o
), displaying an almost Nernstian slope at pH
o
=3. However, when measured during metabolic inhibition,V
m shows no sensitivity to pH
o
over the range 3 to 6, only rising (about 50 mV/pH) at pH
o
=6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1mm of the sugar. Sugar-induced depolarization was insensitive to pH
o
. The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981,J. Membrane Biol.
63:165–190). Three transport systems are characterized: 1) An electrogenic H+ extrusion pump with a stoichiometry of 2 H+ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K+ pathways, there is a passive H+ transport system; here the protonated form seems to be positively charged. 3) A tentative H+-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states. 相似文献
2.
Summary H+-coupled transport in plant and fungal cells is relatively insensitive to external pH (pH
o
). H+-coupled Cl– transport at the plasma membrane ofChara corallina was studied to explore the phenomena responsible for this insensitivity. Raising pH
o
from a control value of 7.5 to 9.0 results in a modest (2.5-fold) decline inJ
max and increase inK
m
. Further increase in pH
o
results in a selective increase inJ
max, in accordance with predictions from a reaction kinetic model of the transport system (Sanders, D., Hansen, U.-P., 1981.J. Membrane Biol.
58:139–153). Increase in cytosolic Cl– concentration ([Cl–]
c
) also results in a selective decrease inJ
max at pH
o
=7.5.Quantitative kinetic modeling of the results is not possible if it is assumed that the sole effect of pH
o
isvia mass action on the binding of external H+ to a transport site. If, instead, the dependence of cytosolic pH (pH
c
) on pH
o
(Smith, F.A., 1984,J. Exp. Bot.
35:1525–1536) is taken into account along with the dependence of Cl– influx on pH
c
(Sanders, D., 1980,J. Membrane Biol.
53:129–141), then the observed modest changes in Michaelis parameters can be accommodated by a reaction kinetic model. The quantitative parameters of the model yield respective pK
a
s of the internal and external H+-binding sites=7.85 and 7.2, respective dissociation constants of the internal and external Cl–-binding sites=160 and 40 m, and an additional, kinetically transparent, H+-binding site with a pK
a
>8.0. The quantitative model independently predicts the response ofJ
max andK
m
to acidic conditions.The results are discussed in terms of the general physiological requirement that fluxes through H+-coupled transport systems are relatively insensitive to environmental variation in pH
o
. It is proposed that (i) the weak (but finite) dependence of pH
c
on pH
o
, coupled with (ii) the strong dependence of H+-coupled transport on pH
c
are instrumental in endowing H+-coupled transport systems with a relative insensitivity to variation in pH
o
. This hypothesis might also explain why pH
c
in plants and fungi is not acutely controlled with respect to variation of pH
o
. 相似文献
3.
Summary We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (H
i
) and external Na+ (Na
o
) in red blood cells of normal subjects.Red blood cells with different cell Na+ (Na
i
) and pH (pH
i
) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pH
i
(from 5.7 to 7.4), external pH (pH
o
), Na
i
and Na
o
and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Na
i
<2.0 mmol/liter cell, Na
o
= 150mm) increased sigmoidally (Hill coefficient 2.5) when pH
i
fell below 7.0 and the external pH
o
was 8.0, but increased linearly at pH
o
6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pH
o
levels (pH
o
8 and pH
o
6). The H+-driven Na+ influx reached saturation between pH
i
5.9 and 6.1. TheV
max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK
m
for H
i
to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 11. The dependence of Na+ influx from Na
o
was studied at pH
i
6.0, and Na
i
lower than 2 mmol/liter cell at pH
o
6.0 and 8.0. The meanK
m
for Na
o
of the H+-gradient-driven Na+ influx was 55±7mm.An increase in Na
i
from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger.It is concluded that human red cell Na+/H+ exchange performs 11 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theV
max between individuals. 相似文献
4.
Summary The intracellular pH (pH
i
) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH
i
of cells placed in an acidic medium (pH
o
below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH
o
is acutely reduced to 5.5, pH
i
falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK
m
is 0.74 ± 0.09 × 10–6
m.Steady-state cells with pH
i
above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH
i
6.3) when returned to pH
o
7.3 medium respond by transporting H+, resulting in a rapid rise in pH
i
. The halftime for this process is 1.09 ± 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH
i
to its steady-state value. 相似文献
5.
N.A. Kuznetsov A.S. Kiryutin A.A. Kuznetsova M.S. Panov M.O. Barsukova 《Journal of biomolecular structure & dynamics》2017,35(5):950-967
Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T)?Tm(εA/T)?Tm(Hx/T)?Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG. 相似文献
6.
Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH
i
=6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pH
o
=6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pH
i
=6.4) markedly increased when pH
o
was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pH0 was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pH
i
was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Na
i
+
=Na
0
+
=15mm,Em=+6.7 mV), an outward H+ gradient (pH
i
=6.4, pH0=8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells. 相似文献
7.
pH
i
recovery in acid-loaded Ehrlich ascites tumor cells and pH
i
maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO
3
–
-free media and at 25 mm HCO
3
–
, the measured pH
i
(7.26 and 7.82, respectively) was significantly more alkaline than the pH
i
. value calculated assuming the transmembrane HCO
3
–
gradient to be equal to the Cl– gradient. Thus, pH
i
in these cells is not determined by the Cl– gradient and by Cl–/HCO
3
–
exchange.pH
i
recovery following acid loading by propionate exposure, NH
4
+
withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3
– free media, and in the presence of HCO
3
–
(25 mm) by DIDS-sensitive, Na+-dependent Cl–/HCO
3
–
exchange. A significant residual pH
i
recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pH
i
regulation. pH
i
maintenance at steady-state involves both Na+/H+ exchange and Na+-dependent Cl–/HCO
3
–
exchange.Acute removal of external Cl– induces a DIDS-sensitive, Na+-dependent alkalinization, taken to represent HCO
3
–
influx in exchange for cellular Cl–. Measurements of 36Cl– efflux into Cl–-free gluconate media with and without Na+ and/or HCO
3
–
(10 mm) directly demonstrate a DIDS-sensitive, Na+
dependent Cl–/HCO
3
–
exchange operating at slightly acidic pH
i
(pHo 6.8), and a DIDS-sensitive, Na+-independent Cl–/HCO
3
–
exchange operating at alkaline pH
i
(pH
o
8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.). 相似文献
8.
S. S. Madsen B. K. Larsen F. B. Jensen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1996,166(2):101-109
Osmoregulation, acid-base balance and respiratory parameters were investigated in whitefish following transfer from freshwater to salt water. Whitefish acclimated successfully to 25 ppt brackish water but died after direct transfer to 32 ppt sea water. Transfer to brackish water induced rapid (<6 h) and permanent increases in plasma [Na+], [Cl–], total [Ca] and [Mg]. The extracellular hyperosmolality effected a transient (<3 days) muscle tissue dehydration and red blood cell shrinkage. Exposure to brackish water decreased both the arterial O2 tension and whole body O2 uptake. The extracellular acid-base status changed from an initial respiratory acidosis at 1 h towards a pronounced metabolic acidosis at 48 h of brackish water exposure. Red cell pHi decreased in parallel with extracellular pHe, but the in vivo pHi/pHe was only 0.26, suggesting some selective protection of red cell pHi. Plasma cortisol concentration and gill Na+, K+-ATPase activity increased after exposure to high ambient salinity, reflecting the induction of hypo-osmoregulatory mechanisms. The physiological changes in whitefish are discussed in relation to salinity-induced effects in other salmonid fishes.Abbreviations
CO2 solubility in plasma
-
water O2 capacitance coefficient
-
BW
brackish water
-
C
T
total CO2 content in plasma
-
FW
fresh water
-
Hb
hemoglobin
-
Hct
hematocrit
-
M
b
body mass of fish
-
MCHC
mean cellular hemoglobin concentration
-
PCO2
carbon dioxide tension
-
pH
e
extracellular pH
-
pH
i
intracellular pH
-
PO2 in
oxygen tension in water flowing in
-
PO2 out
oxygen tension in water flowing out
-
ppt
parts per thousand
-
RBC
red blood cell(s)
-
SW
sea water
-
V
m
water flows through chamber
-
OV
2
ml O2 consumed per kg per hour 相似文献
9.
Davis BA Hogan EM Cooper GJ Bashi E Zhao J Boron WF 《The Journal of membrane biology》2001,183(1):25-32
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular
alkali loads by moving K+ and HCO−
3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO−
3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO−
3 influx (J
HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl−. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl−. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO−
3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO−
3. With no QA+ in the DF, J
HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J
HCO3 was virtually zero. The apparent K
i
for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J
HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K
i
value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K
i
of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
Received: 21 November 2000/Revised: 14 May 2001 相似文献
10.
Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might
play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH
(pH
i
) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived
from rabbit NPE. pH
i
was measured using the fluorescent dye BCECF and the pH
i
responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid
pH
i
changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and
inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH
i
. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mm) reduced pH
i
0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH
i
reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH
i
reduction. DBI also reduced by ∼40% the rate of pH
i
recovery in cells acidified by an ammonium chloride (20 mm) prepulse; a reduction in pH
i
recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH
i
alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive
to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We
suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO−
3 with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH
i
changes that might alter the function of other ion transporters and channels in the NPE.
Received: 24 April 1997/Revised: 4 November 1997 相似文献
11.
The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH
4
+
and high concentrations of K+ on cytoplasmic pH (pH
i
) were investigated in dispersed and cultured pancreatic -cells fromob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pH
i
value in resting -cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pH
i
with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pH
i
. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pH
i
. In contrast to D-glucose, 10mM of D-glyceraldehyde decreased pH
i
by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pH
i
in pancreatic -cells. 相似文献
12.
Ira Kurtz 《The Journal of membrane biology》1988,106(3):253-260
Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH
i
) was measured using BCECF. Steady-state pH
i
in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na+, pH
i
decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH
i
was significantly less, 0.73±0.18 pH/min. Steady-state pH
i
decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na+ 140mm lumen and bath). When Na+ was removed from the basolateral side of the tubule, pH
i
decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH
i
was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na+ 140mm lumen and bath) caused steady-state pH
i
to decrease significantly by 0.06 pH units. When pH
i
was acutely decreased to 5.87±0.02 following NH4Cl removal (lumen, bath), pH
i
failed to recover in the absence of Na+ (lumen, bath). Addition of 140mm Na+ to the lumen caused pH
i
to recover at a rate of 2.17±0.59 pH/min. The rate of pH
i
recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na+ was added to the basolateral side, pH
i
recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH
i
, by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na+/N+ antiporters. In the steady state, the rate of Na+-dependent H+ flux across the apical antiporter exceeds the rate of Na+-dependent H+ flux via the basolateral antiporter. Recovery of pH
i
following acute intracellular acidification is Na+ dependent and mediated primarily by the luminal antiporter. 相似文献
13.
Charles M. Schron 《The Journal of membrane biology》1990,118(3):259-267
Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest.
76:2030–2033), suggesting carrier-mediated folate: OH– exchange (or phenomenologically indistiguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pHint/pHext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+
K
m
=12.8±1.2m). Hill plots of these data suggest involvement of at least one H+ (OH–) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F– and F–2 is electroneutral. Kinetic parameters for F– and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pHext 5.8 (K
m
=1.33vs. 1.70 m;V
max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV
max, but does not affect thatK
m, for carrier-mediated F transport. These data are consistent with similarK
i's for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H+
K
m
(or OH– IC50) corresponding to pH 4.89. External pH affects theV
max, but not theK
m
for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH– or H+) rather than competitive binding that is mutually exclusive. 相似文献
14.
Hans Oberleithner Ulrich Kersting Stefan Silbernagl Wieland Steigner Ulrich Vogel 《The Journal of membrane biology》1989,111(1):49-56
Summary We have chosen the MDCK cell line to investigate aldosterone action on H+ transport and its role in regulating cell membrane K+ conductance (G
m
K
). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K+ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH
i
) andG
m
K
were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH
i
andG
m
K
(Hill coefficient 4.4±0.4), indicating multiple H+ binding sites at a single K+ channel. Application of aldosterone increased pH
i
within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO2/HCO
3
–
buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO2/ HCO
3
–
is limited by the increased activity of a pH
i
-regulating HCO
3
–
extrusion system. SinceG
m
K
is stimulated half-maximally at the pH
i
of 7.18±0.04, internal H+ ions could serve as an effective intracellular signal for the regulation of transepithelial K+ flux. 相似文献
15.
Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises Tm, the melting temperature of free base pairs. The linear dependence of Tm on log Na+ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na+ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs Tm′ remains constant at various ionic strengths. On the other hand, the Tm′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of Tm′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na+ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers Tm′. This explanation is supported by circular dichroism (CD) results. 相似文献
16.
《Journal of thermal biology》1999,24(3):199-208
A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pHi) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C (Notothenia coriiceps; Antarctica), 5°C (Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C (Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pHe) did not follow the constant relative alkalinity relationship, where pH+=pOH− for any particular temperature, over a range of 1–26°C (overall δpHe/δT=0.009±0.002 U °C−1; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pHi) was also regulated independently of pN(=0.5 pK water) in all species of fish examined. The inverse relationship between pHi and environmental temperature gave an overall δpHi/δT of −0.010±0.001 U °C−1 (for both white and red muscle) and −0.004±0.003 U °C−1 (cardiac muscle). However, between 1 and 11°C δpHi/δT was much higher (P<0.001), −0.022±0.003 U °C−1 (white muscle) and −0.022±0.004 U °C−1 (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pHi, are discussed. 相似文献
17.
Double-helical poly(dG-dC) and poly(dA-dT) are DNA analogs in which the interactions between the two strands of the helix are, respectively, either the stronger G/C type or the weaker A/T type along the entire length of macromolecules. Thus, these synthetic polynucleotides can be considered as representatives of the most stable and the least stable DNA. In the investigations presented here, potentiometric titrations and stopped-flow kinetic experiments were carried out in order to compare the pH-induced helix–coil conformations (10°C and 150mM [Na+]) the pH of the helix–coil transition (pHm) is 12.81 for poly(dG-dC) and 11.76 for poly(dA-dT). The unwinding of double-helical poly(dG-dC) initiated by a sudden change in pH was found to be a simple exponential process with rate constants in the range of 200–600 sec?1, depending on the final value of the pH jump. The intramolecular double-helix formation of poly(dG-dC) was studied by lowering the pH of the solutions from a value above pHm to that below pHm in dilute solutions (15.5 ug/ml [polymer]). Under these conditions, the observed rewinding reactions displayed a major and two exponential phases, all of which were independent of polymer concentration. From the comparison of the results of poly(dA-dT) and poly(dG-dT) would unwind faster than poly(dG-dC). However, if the pH jumps are such that they present the same perturbation of these polymers relative to their pHm values, no significant differences exist between the rates of helix–coil conformation changes of poly(dA-dT) and poly(dG-dC). 相似文献
18.
R. Aaron Rogers Madeline R. Meyer Kayla M. Stewart Gabriela M. Eyring Aaron M. Fleming Cynthia J. Burrows 《Biopolymers》2021,112(1)
In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values. 相似文献
19.
S. Gaillard J. L. Rodeau 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1987,157(4):435-444
Summary Intracellular pH (pHi) regulation was studied in crayfish neurons with pH-, and Na+-sensitive microelectrodes. It was confirmed to involve both a HCO
3
–
-dependent and a HCO
3
–
-independent mechanism. The latter was identified as the amiloride-sensitive Na+/H+ exchange described in vertebrate cells. Its dependence on extracellular pH (pHe) and Na+ concentration ([Na+]e) was studied in CO2-free external solutions at 20°C. The steady state pHi and the rate constant (k) of the exponential pHi recovery following an acid load were determined. At pHe=7.5 and [Na+]e=200 mM, the average steady state pHi was 7.09±0.12 (as compared to 7.30±0.10 in the presence of 5 mM bicarbonate). The dependence of the rate constant of recovery on [Na+]e could be described by Michaelis-Menten kinetics; at pHe=7.5 the apparentK
m andK
max were 39 mM and 1.4 mmol·l–1·min–1, respectively. Decreasing pHe reduced the rate of recovery, the variations ofk with pHe conforming to a simple titration curve with an apparent pK of 7.05±0.21. These kinetic properties of the Na+/H+ exchange in crayfish neurons are similar to those described in vertebrate cells.Preliminary results were presented at the First International Congress of Comparative Physiology and Biochemistry (Liège, Belgium, 1984) 相似文献
20.
《Nucleosides, nucleotides & nucleic acids》2013,32(5-8):573-576
Abstract The pyrazolo[3,4-d]pyrimidine-4,6-diamine nucleosides 2b-d stabilize the dA-dT base pair significantly when the dA-residue is replaced. Oligonucleotide duplexes incorporating 2b-d show a 4–6°C T m increase per modification. The 7-bromo compound 2b harmonizes the stability of the dA-dT vs. the dG-dC pair. According to this the stability of such duplexes depends no longer on the base pair composition of a DNA molecule. 相似文献