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1.
Effect of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodo-l-thyronine (T3) or replacement therapy (TR) with T3 + l-thyroxine (T4) on the temperature kinetics properties of FoF1 adenosine triphosphatase (ATPase, ATP synthase, H+-translocating ATP synthase EC 3.6.3.14)
and succinate oxidase (SO) and on the lipid/phospholipid makeup of rat kidney mitochondria were examined. Tx lowered ATPase
activity, which T3 treatment restored. SO activity was unchanged in Tx but decreased further by T3 treatment. TR restored both activities. The energies of ATPase activation in the high and low temperature ranges (E
H and E
L) increased in the Tx and T3 animals with decrease in phase transition temperature (Tt). TR restored E
H and E
L but not Tt to euthyroid levels. E
H and E
L of SO decreased in Tx animals. T3 and TR restored E
H whereas E
L was restored only in the TR group; Tt increased in both groups. Total phospholipid and cholesterol contents decreased significantly in Tx and T3-treated animals. In Tx animals, sphingomyelin (SPM) and phosphatidylcholine (PC) components decreased, while phosphatidylserine
(PS) and diphosphatidylglycerol components increased. T3 and TR treatments caused decreases in SPM, phosphatidylinositol and PS. PC and phosphatidylethanolamine (PE) increased in the T3 group. TR resulted in increased lysophospolipids and PE. Changes in kinetic parameters of the two enzymes were differently correlated
with specific phospholipid components. Both T3 and TR regimens were unable to restore normal membrane structure-function relationships. 相似文献
2.
Summary The apparent energy of activation (E
a), Michaelis-Menten constant (K
mfor oxaloacetate), V
max/K
mratios and specific activities of NADP+-malate dehydrogenase (NADP+-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO2 concentrations, 350 l l-1 and 675 l l-1. E
avalues of NADP+-MDH extracted from QUE plants were significantly lower than those of MISS plants. K
mvalues and V
max/K
mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V
max/K
mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO2 enrichment did not modify appreciably the catalytic properties of NADP+-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP+-MDH activities were always in excess of the amount required to support observed rates of CO2 assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP+-malate dehydrogenase among these ecotypes. 相似文献
3.
Summary We report a study of a potassium-selective channel in the membrane delineating cytoplasmic drops fromChara australis. The relatively large conductance (170 pS in 150 mol/m3 (mm) KCl), high ion selectivity (P
Cl/P
K=0.015±0.01) and voltagedependent kinetics of this channel indicate that it is a type of maxi-K channel commonly found in animal cells but not previously detected in any plant cell.The current-voltage (I/V) characteristic of these channels was examined in drop-attached and in excised outside-out patches using the patch-clamp technique, over the unusually large voltage range of –250 to 200 mV. TheI/V characteristic is nonlinear and shows saturation at extreme voltages; the current also saturates at high [K+]. In solutions with symmetrical KCl concentrations the saturation behavior of the current is asymmetrical. The permeability of the channel depends on whether it is observed in excised or in drop-attached membrane patches.Here we investigate the main factors affecting the permeation of K+ ions through this maxi-K channel. We present the first direct evidence for the importance of diffusion external to the pore in limiting ion flow through maxi-K channels. The data are consistent with an ion translocation mechanism whose current is limited (i) at high voltages by ion diffusion external to the pore and (ii) at high [K+] by the maximum transport rate of the channel. We fit the data to a diffusion-limited pore model in which the pore exhibits saturation described by Michaelis-Menten kinetics with aK
m=50±25 mol/m3 andG
max=300±20 pS. 相似文献
4.
The kinetics of NH4
+ and NO3
− uptake in young Douglas fir trees (Pseudotsuga menziesii [Mirb.] Franco) were studied in solutions, containing either one or both N species. Using solutions containing a single N
species, the Vmax of NH4
+ uptake was higher than that of NO3
− uptake. The Km of NH4
+ uptake and Km of NO3
− uptake differed not significantly. When both NH4
+ and NO3
− were present, the Vmax for NH4
+ uptake became slightly higher, and the Km for NH4
+ uptake remained in the same order. Under these conditions the NO3
− uptake was almost totally inhibited over the whole range of concentrations used (10–1000 μM total N). This inhibition by NH4
+ occurred during the first two hours after addition. ei]{gnA C}{fnBorstlap} 相似文献
5.
Bent Vestergaard-Bogind Per Stampe Palle Christophersen 《The Journal of membrane biology》1987,95(2):121-130
Summary The conductance of the Ca2+-activated K+ channel (g
K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V
m
) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV
m
, monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E
K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V
m
–E
K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V
m
–E
K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V
m
–E
K) decreased from 20 towards zero mVg
K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV
m
(–50 mV to 0). A further increase ing
K(Ca) symmetrically aroundE
K is suggested as (V
m
–E
K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing
K(Ca) from 50 to 70 S/cm2. Since the driving force (V
m
–E
K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg
K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel. 相似文献
6.
Summary The effects of short- and long-term exposure to a range in concentration of sea salts on the kinetics of NH
inf4
sup+
uptake by Spartina alterniflora were examined in a laboratory culture experiment. Long-term exposure to increasing salinity up to 50 g/L resulted in a progressive increase in the apparent Km but did not significantly affect Vmax (mean Vmax=4.23±1.97 mole·g–1·h–1). The apparent Km increased in a nonlinear fashion from a mean of 2.66±1.10 mole/L at a salinity of 5 g/L to a mean of 17.56±4.10 mole/L at a salinity of 50 g/L. These results suggest that the long-term effect of exposure to total salt concentrations within the range 5–50 g/L was a competitive inhibition of NH
inf4
sup+
uptake in S. alterniflora. No significant NH
inf4
sup+
uptake was observed in S. alterniflora exposed to 65 g/L sea salts. Short-term exposure to rapid changes in salinity significantly affected both Vmax and Km. Reduction of solution salinity from 35 to 5 g/L did not change Vmax but reduced Km by 71%. However, exposing plants grown at 5 g/L salinity to 35 resulted in an decrease in Vmax of approximately 50%. Exposure of plants grown at 35 g/L to a total sea salt concentration of 50 g/L for 48h completely inhibited uptake of NH
inf4
sup+
. For both experiments, increasing salinity led to an increase in the apparent Km similar to that found in response to long-term exposure. Our data are consistent with a conceptual model of changes in the productivity of S. alterniflora in the salt marsh as a function of environmental modification of NH
inf4
sup+
uptake kinetics. 相似文献
7.
With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na+ + K+)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK
m is 2 orders of magnitude less and the Vmax is two times greater, and dimethyl sulfoxide (Me2SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK
m and Vmax for K+-activated 3-OMFP hydrolysis as well as theK
0.5 for K+ activation. However, reducing the incubation pH increases inhibition by Pi and the Vmax for 3-OMFP hydrolysis in the absence of K+. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K+-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K+, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na+, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na+ + K+)-phosphatase pathway, most apparent at suboptimal K+ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et3N
triethyl amine
- FITC
fluorescein isothiocyanate
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonate
- MES
2-(N-morpholino)ethanesulfonate
- Me2SO
dimethyl sulfoxide
- NPP
p-nitrophenyl phosphate
- 3-OMFP
3-O-methylfluorescein phosphate
- TNP-ATP
2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP 相似文献
8.
Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K
m
= 3 m and low capacity (Vmax) 0.4 mol l-Glu · Kg–1 dry cell mass · 10 min–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X
AG
–
family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K
m 39 m but high capacity (V
max) 1.8 mol l-Glu · Kg–1 dry cell mass · 10 min–1. 相似文献
9.
K. Hager A. Hazama H. M. Kwon D. D. F. Loo J. S. Handler E. M. Wright 《The Journal of membrane biology》1995,143(2):103-113
The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V
m
), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: K
0.5
MI
, K
0.5
Na
, and the Hill coefficient n. At 100 mM NaCl, K
0.5
MI
was about 50 m and was independent of V
m
. At 0.5 mm
myo-inositol, K
0.5
Na
ranged from 76 mm at V
m
=–50 mV to 40 mm at V
m
=–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K
I
of 64 m at V
m
=–50 mV and 130 m at V
m
= –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V
m
=–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V
m
=–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na+-dependent uptake of 3H-d-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na+ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V
m
. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812
Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554. 相似文献
10.
Michael R. Blatt 《The Journal of membrane biology》1988,102(3):235-246
Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K+, fluxes thought to pass through K+ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K+ channels, its pharmacology and dependence on external K+ (K
v
+
) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V
m
) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K+ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K+ current rose with voltage positive toV
m
but it decayed over two voltage ranges, one negative toV
m
and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K
v
+
and followed the K+ equilibrium potential. Against a (presumably) low background of primary ion transport, the K+ current contributed appreciably to charge balance atV
m
in 0.1mm as well as in 1 to 10mm K
v
+
. Thus, gating of these K+ channels compensates for the prevailing K+ conditions to ensure net K+ movement out of the cell. 相似文献
11.
Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK
m
over 120-fold but only decreases theV
max fourfold. SuchK
m
mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV
max 80-fold without changing theK
m
.V
max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK
m
andV
max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport. 相似文献
12.
Summary Human red cells were prepared with various cellular Na+ and K+ concentrations at a constant sum of 156mm. At maximal activation of the K+ conductance,g
K(Ca), the net efflux of K+ was determined as a function of the cellular Na+ and K+ concentrations and the membrane potential,V
m
, at a fixed [K+]ex of 3.5mm.V
m
was only varied from (V
m
–E
K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g
K(Ca) as a function of cellular Na+ and K+ concentrations atV
m
=–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na+. Since the present Ca2+-activated K+ channels have been shown to be of the multi-ion type, the experimental data from each set of Na+ and K+ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na+ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na+ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca2+ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg
K(Ca) for outward currents at (V
m
–E
K)>25 25 mV reflects a voltage-dependent Na+ block of the Ca2+-activated K+ channels. 相似文献
13.
Missan S Zhabyeyev P Dyachok O Ogura T McDonald TF 《The Journal of membrane biology》2004,202(3):151-160
Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K+ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K+ current (IK1), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrodes solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3–2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba2+-sensitive IK1, an increase in the amplitude of outward IK1, and a steepening of the slope of the inward IK1-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (GK1) and rectification, GK1-V data were fitted with Boltzmann functions to determine maximal GK1 (GK1max) and voltage at one-half GK1max (V0.5). Superfusion with hyperosmotic sucrose solutions led to significant increases in GK1max (e.g., 28±2% with 1.8T), and significant negative shifts in V0.5 (e.g., –6.7±0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that GK1max and V0.5 were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K+. We conclude that the effects of hyperosmotic sucrose solutions on IK1 are related to shrinkage-induced concentrating of intracellular K+. 相似文献
14.
The efficacy and mechanism of -dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mm K
o
+
. However, DTX block was dependent on the external K+ concentration. Raising K+ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K
i
= 10 mm in 10 nm DTX). The effect of K+ in protecting I
K,in
was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a K
d
for DTX of 240 pm. A K
d
of 410 m for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 1 mm) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship. 相似文献
15.
Apparent half-saturation constants (K
m) and maximum uptake rates (V
max) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a35S-sulfate tracer technique. The lowerstK
m (5 M) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestK
m (77 M) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,V
max) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsK
m andV
max between 1 and 6,000 M SO
4
2-
, and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.Abbreviations MPB
methane producing bacteria
- SRB
sulfate reducing bacteria 相似文献
16.
Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V
m
) and intracellular K+ activity (a
K
i
) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V
m
increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV
m
/controlV
m
)] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea
K
i
did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K+ or 1mm quinine HCl or at 27°C resulted in no change inV
m
compared with a 20-mV increase inV
m
without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda
K
i
in response to anisosmotic medium; however,V
m
behaves as an osmometer under these conditions. Also, increases inV
m
by hypoosmotic stress were abolished by conditions or agents that inhibit K+ conductance. 相似文献
17.
Cavarra MS del Mónaco SM Assef YA Ibarra C Kotsias BA 《The Journal of membrane biology》2007,219(1-3):49-61
The human ether-a-go-go related gene (HERG1) K+ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or
apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally
truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments.
Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization
to voltages negative to −40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel
blocker, E4031. Half-maximal inhibitory concentration (IC50) of the blocker was 4.69 nm. The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current,
known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at −120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with
some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown
to increase HERG1a K+ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell
currents were not activated by H2O2. 相似文献
18.
Marshall H. Montrose Geraldine Bebernitz George A. Kimmich 《The Journal of membrane biology》1985,88(1):55-66
Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H+ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K+ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H+ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na+/H+ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na+ gradient. The kinetics of the Na+/H+ exchange reaction at pH 6.0 [K
t
for Na+=57mm,V
max=42 mmol H+/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K
t
for Na+=15mm,V
max=1.7 mmol H+/liter 3OMG space/min). Experiments involving imposed K+ gradients suggest that these cells have negligible K+/H+ exchange capability. They exhibit limited but measurable H+ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate. 相似文献
19.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
20.
Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na+-K+-ATPase activity from the dihydroouabain-sensitive current (I
DHO) in the presence of increasing concentrations of tetraethylammonium (TEA+; 0, 5, 10, 20, 40 mm), a well-known blocker of K+ channels. The effects of TEA+ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V
M) and a saturable inhibitory component affecting an outward current easily detectable at positive V
M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA+ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA+ on K+ channels. Interestingly, this latter component disappears when the Na+-K+-ATPase is inhibited by 10 m DHO. Conversely, TEA+ inhibits a component of I
DHO with a k
d of 25±4 mm at +50 mV. As the TEA+-sensitive current present in I
DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K+ channels whose activity varies in parallel with the Na+-K+-ATPase activity. Supporting this hypothesis, the inward portion of this TEA+-sensitive current can be completely abolished by the addition of 1 mm Ba2+ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA+-sensitive K+ currents and indicates that, in the absence of effective K+ channel inhibitors, I
DHO does not exclusively represent the Na+-K+-ATPase-generated current. 相似文献