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1.
To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.  相似文献   

2.
3.
The activity coefficients of glycylglycine in four aqueous electrolyte solutions (+NaCl, +NaBr, +KCl and +KBr) were obtained at 298.2 K. The mean ionic activity coefficient of the electrolyte in aqueous solutions containing the peptide was determined from measurements of the potential differences of a cation and an anion ion-selective-electrode, each vs. a double junction reference electrode. The results show that the nature of the anion has a major effect on the activity coefficients of glycylglycine. Comparison of activity coefficient data for glycylglycine with literature data for glycine, both in aqueous NaCl solutions, indicates that the effect of the electrolyte is larger for the peptide than for the amino acid. For the peptide, in all cases, the effect of the electrolyte is more important at low molalities of the electrolyte. The Wilson equation was used to correlate the activity coefficient data obtained. The correlation results were satisfactory for the region of concentrated electrolyte.  相似文献   

4.
  • 1.1. Membrane-bound (Na+ + K+)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P3) of the electrolyte.
  • 2.2. The effect of Li+ and Ba2+ on (Na+ + K+)-ATPase activity of the two membrane fractions (P2 and P3) was analysed with respect to K+ and Mg2+ ions, after the I50 estimation.
  • 3.3. The kinetics of the reactions with these cations were investigated showing that Li+ inhibits P2 uncompetitively and for P3 presented a mixed type inhibition.
  • 4.4. Ba2+ behaved as an hyperbolic mixed type inhibitor for P2 and a linear mixed type inhibitor for P3 fraction.
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5.
We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.  相似文献   

6.
The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.  相似文献   

7.
Danilo Guerini 《Biometals》1998,11(4):319-330
The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers  相似文献   

8.
The influence of H+ and K+ on the partial reactions and transport of gastric (H+ + K+)-ATPase was studied. Using transient kinetics, the effects and sidedness of effects of H+ and K+ on formation and breakdown of phosphoenzyme were determined in intact and lyophilized reconstituted vesicles in the absence and presence of gramicidin. Whereas increasing H+ concentrations on the ATP-binding face of the vesicles accelerates phosphorylation, increasing K+ concentrations inhibits phosphorylation. Increasing H+ on this side reduces K+ inhibition of the phosphorylation rate. At low ATP/K+ ratios, the phosphorylation step can become rate-limiting for steady state hydrolysis. Decreasing H+ accelerates dephosphorylation in the absence of K+. K+ on the internal or luminal face of the vesicles accelerates dephosphorylation, and this rate is reduced with increasing H+ concentrations. At low internal pH, K+-dependent dephosphorylation may become rate-limiting. H+ transport measurements using fluorescence quenching of acridine orange show that whereas internal K+ is required for H+ transport, external K+ inhibits the rate of formation of a pH gradient, and the inhibition is reduced by decreasing medium pH. The pH optimum for ATPase activity and transport correlated in the vesicles, and the K0.5 of K+ for transport correlated with data for intact parietal cells.  相似文献   

9.
The effect of TNF-alpha on the renal Na+-K+ pump and the Na+-K+2Cl- cotransporter was investigated in the rat. Animals were injected with the cytokine, and 4h later, a homogenate from the cortical and medullary tissues was prepared and used to assay the activity of the Na+-K+ ATPase and the protein expression of the pump and symporter. TNF-alpha reduced the activity and expression of the pump in both cortex and medulla, and its effect disappeared when animals were pre-treated with indomethacin, suggesting that TNF-alpha acts via PGE2. Higher levels of PGE2 were detected by enzyme immunoassay, in kidney tissues isolated from rats treated with PGE2, thus confirming this hypothesis. The cytokine also down-regulated the Na+-K+2Cl- cotransporter but this effect was not abrogated by indomethacin. PGE2, injected into animals, exerted a dose-dependent effect. Low doses did not have any effect on the two transporters in the cortex while high doses inhibited and down-regulated the pump and up-regulated the cotransporter. In the medulla low doses increased the activity and expression of the pump but down-regulated the cotransporter while high doses exerted an exactly opposite effect on the two transporters. It was concluded that the effect of TNF-alpha on the pump is mediated via PGE2 which is released at relatively high doses. The effect of the cytokine on the cotransporter is, however, independent of PGE2.  相似文献   

10.
Using the compartmental analysis the unidirectional Na+ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa+ contents Qc and Qv, and the trans-root Na+ transport R'have been studied as a function of the external Na+ concentration.Using the re-elution technique the effect of low K+ concentrationson the plasmalemma efflux co of Na+ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K+-Na+exchange system. The apparent Km corresponded to a cytoplasmicNa+ concentration of 28 mM at 0·2 mM K+ and about 0·2mM Na+ in the external solution. 0·2 mM K+ stimulatedthe plasma-lemma efflux of Na+ and inhibited Na+ transport selectivelyeven in the presence of 10 mM Na+ in the external medium showingthe high efficiency of the K+-Na+ exchange system. However,co, K+-dep was inhibited at 10 mM Na1 compared to lower Na1concentrations suggesting some competition of Na1 with K1 atthe external site of the exchange system. The effect of theNa+ concentration on Na1 influx oc is discussed with respectto kinetic models of uuptake.  相似文献   

11.
The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).  相似文献   

12.
The rate of the transition from the E2 form to the E1 form of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been monitored by the fluorescence changes of eosin. The equilibrium between E1 and E2 is poised towards E2 in the absence of added cations. A stopped-flow tracing of the transition from E2 in the presence of 2 microM K+ (contamination) to E1 (in 150 mM Na+) is multiexponential with a large, rapidly decaying component (t 1/2 about 50 ms) and a smaller component which has a t 1/2 of about 2 s. KCl in microM concentrations decreases the amplitude of the rapidly decaying component and increases the amplitude of the slow component. The stopped-flow tracings can be satisfactorily fitted by a sum of three exponentials. An apparent Kd for K+ of about 5 microM is obtained for the conversion of the rapidly decaying component to the slowly decaying component. The experiments show that the E2 form is a mixture of at least two enzyme conformations. One E2 conformation - without K+ bound, (E2) - is transferred rapidly to the E1 conformation when Na+ is added, whereas the other E2-conformation--with K+ bound with an apparent high affinity, Kocc E2--is transferred slowly to the E1 conformation.  相似文献   

13.
L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.  相似文献   

14.
In the absence of Na+ in the medium, the membrane potential of obligately alkalophilic Bacillus cells was found to be decreased by the addition of K+ to the medium, whereas K+ addition in the presence of Na+ had no effect. Rb+ showed essentially the same effect as K+. The decreased membrane potential was quickly restored by lowering the K+ concentration in the medium or by adding Na+ or Li+ to the medium. Thus, in the absence of Na+, the membrane potential of alkalophilic Bacillus seems to be affected by the concentration difference of K+ between inside and outside of the cell, and Na+ or Li+ in the medium suppresses the K+ effect. An exchange between extracellular Rb+ and intracellular K+ was observed in the absence of Na+. However, the exchange was greatly suppressed by the addition of Na+ or Li+ to the medium, indicating that Na+ in the medium modulates the K+ permeability of the alkalophilic Bacillus cell membrane. The K+-induced decrease in the membrane potential of alkalophilic Bacillus in the absence of Na+ is accounted for by the increased K+-permeability of the cell membrane.  相似文献   

15.
TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.  相似文献   

16.
The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.  相似文献   

17.
We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic structural homology between the retinal Na+-Ca2+, K+ and the cardiac Na+-Ca2+ exchange proteins. Sets of mAbs were raised separately to partially purified preparations of either the retinal or the recombinant myocardial exchanger. Each panel of mAbs was then screened for crossreactivity with the respective heterologous exchanger using enzyme-linked immunoassay and immunoblotting techniques. Out of 43 anti-retinal exchanger mAbs, we found 3 detecting the cardiac exchanger on immunoblots, while 4 out of 36 anti-cardiac exchanger mAbs reacted with the retinal exchanger. The strength of the crossreactions was generally weak and suggested that only low affinity epitopes were available on the heterologous proteins. For two crossreacting anti-retinal mAbs the apparent binding affinities to the cardiac exchanger were lower by more than two orders of magnitude. The overall low degree of epitope sharing among the two sets of mAbs confirms that in spite of their obvious functional and topological similarities, microscopic structural homologies between the two proteins are scarce.  相似文献   

18.
1. The major ionmotive ATPase, in animal cells, is the Na+, K+-ATPase or sodium pump.2. This membrane bound enzyme is responsible for the translocation of Na+ ions and K+ ions across the plasma membrane, an active transport mechanism that requires the expenditure of the metabolic energy stored within the ATP molecule.3. This ubiquitous enzyme controls directly or indirectly many essential cellular functions, such as, cell volume, free calcium concentration and membrane potential.4. It is, therefore, apparent that alterations in its regulation may play key roles in pathological processes.  相似文献   

19.
MthK is a Ca2+-gated K+ channel whose activity is inhibited by cytoplasmic H+. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H+ inhibition and Ca2+-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (ranging from nominally 0 to 30 mM) at a given [H+], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of [H+] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) and log([H+]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H+ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+.  相似文献   

20.
Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa+-K+pump for Na+. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa+. When pipette solutions wereK+ free, pump current(Ip) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in Ipmeasured with pipettes containingK+. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in Ip wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofIp. We concludethat ANG II regulates the pump's selectivity for intracellularNa+ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade.

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