共查询到20条相似文献,搜索用时 15 毫秒
1.
Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes 总被引:2,自引:0,他引:2
Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell. 相似文献
2.
Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter 总被引:2,自引:2,他引:0
《The Journal of general physiology》1993,101(6):889-908
To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
3.
Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell. 相似文献
4.
Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme 总被引:18,自引:0,他引:18
Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme. 相似文献
5.
(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+ 总被引:4,自引:0,他引:4
Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake. 相似文献
6.
Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange. 相似文献
7.
This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains. 相似文献
8.
J S Wiley R Chen M J Wiley G P Jamieson 《Archives of biochemistry and biophysics》1992,292(2):411-418
Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs. 相似文献
9.
Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells 总被引:8,自引:0,他引:8
[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes. 相似文献
10.
Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP 总被引:2,自引:0,他引:2
B Forbush 《The Journal of biological chemistry》1987,262(23):11104-11115
We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle. 相似文献
11.
Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate 总被引:1,自引:0,他引:1
B Forbush 《The Journal of biological chemistry》1987,262(23):11116-11127
The rate of 86Rb or 42K release from an occluded form of the phosphorylated Na+ pump has been studied using a rapid filtration apparatus described previously. The rate constant of release is 5-15 s-1, and 42K and 86Rb dissociate at approximately the same rate. Mg2+ is required for deocclusion in the presence of Pi at a site which has the same affinity as the site involved in stabilization of E2(K) with ATP; we propose that Na,K-ATPase has only one site for Mg2+ (apart from Mg2+ complexed with ATP), that the affinity of this site for Mg2+ is increased by Pi binding and decreased by ATP binding, and that Mg2+ is bound and released in the normal transport cycle. In the presence of K+, Cs+, Rb+, or Tl+, the release of two distinct 86Rb ions can be observed, the slow release from one site ("s" site) being blocked by occupancy of the site vacated by the other ("f", fast site). By a sequence of incubations, labeled 86Rb can be placed at either site, and the rate of dissociation monitored individually; in the absence of K+, dissociation from the s site proceeds after a lag in which the f site is vacated. The results are consistent with a "flickering-gate" model of deocclusion to the extracellular pump face, in which the site is exposed to the medium only long enough for a single ion to be released. When deocclusion to the intracellular face is promoted with ATP, ions are released from both sites at the same rate, presumably because the E2----E1 conformational change is rate-limiting. Unlabeled ions co-occluded with 86Rb increase the ATP-stimulated rate of release in the order Rb+ less than Tl+ less than Cs+ less than K+; since the same rank order is observed when dissociation from the s site is monitored in the presence of these ions and MgPi we propose that the latter process proceeds toward the intracellular pump face. 86Rb release from the vanadate-inhibited enzyme has the characteristics of Pi-stimulated release but is approximately 25-fold slower. ATP binds to both the phosphorylated and vanadate-inhibited forms of Na,K-ATPase and increases the rate of deocclusion, apparently to both the intracellular and extracellular faces of the pump. 相似文献
12.
The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump. 相似文献
13.
R Blostein 《The Journal of biological chemistry》1983,258(20):12228-12232
Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting. 相似文献
14.
Sluyter R Shemon AN Barden JA Wiley JS 《The Journal of biological chemistry》2004,279(43):44749-44755
Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor. 相似文献
15.
B Forbush 《The Journal of biological chemistry》1988,263(17):7979-7988
We have studied the effect of various amines on the rate of release of 86Rb from the occluded state of dog kidney Na,K-ATPase formed by pre-incubation of the enzyme with 86Rb. In the presence of MgPi, various amines act like K+ or Rb+ in blocking the release of 86Rb from one of two sites for occlusion (the "s" site). Of 38 amines tested, tetrapropylamine and various benzyl amines exhibit the highest affinity; the K1/2 for these compounds is 2-5 mM. In the presence of ATP, when 86Rb is presumably released towards the intracellular face of the pump in the normal mode of operation, 86Rb release is blocked by the presence of amine, but only if the amine is also included in a preincubation with MgPi. The data are consistent with a model in which the interaction of amine with one of the transport sites (the "f" site) prevents the E2----E1 transformation that is stimulated by ATP. When 86Rb deocclusion from the f site has occurred in the presence of amine, the lone 86Rb at the s site can be released in the presence of ATP if the amine is removed from the medium. This suggests that a single 86Rb ion at the s site can be released to the intracellular face of the membrane, and therefore that transport can occur with only one K+ site occupied. The amine that blocks release of one 86Rb ion does not itself become occluded: (a) The interaction of amine and ATP is only seen when both ligands are present in the medium; (b) the effects of amines are not "remembered" after a brief exposure to a rinse medium; (c) with the vanadate-inhibited enzyme, benzyltriethylamine and tetrapropylamine are only weakly effective in blocking 86Rb release from the s site; and (d) organic cations exhibit very low affinity in competition with 86Rb for occlusion at equilibrium. Thus the results are consistent with the idea that monofunctional amines block by binding to the f site but that, unlike K+ and Rb+, they do not become occluded. In contrast, at equilibrium ethylenediamine prevents 86Rb occlusion in a competitive manner, suggesting the possibility of occlusion of the bifunctional amine. 相似文献
16.
K Satoh T Nakao F Nagai I Kano A Nakagawa K Ushiyama O Urayama Y Hara M Nakao 《Biochimica et biophysica acta》1989,994(2):104-113
Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles. 相似文献
17.
Rolando C. Rossi Segio B. Kaufman Rodolfo M. Gonzlez Lebrero Jens G. Nrby Patricio J. Garrahan 《Analytical biochemistry》1999,270(2):276-285
The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactants in particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25 degrees C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15-40 ml s-1. Quenching requires that the process studied is slowed considerably by cooling to 0-2 degrees C and/or by removal of reactants by dilution. The equipment was characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22-0.80 microm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb+ was formed in the RMA under conditions where the rate constant for release of Rb+ at 25 degrees C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3-4 ms. 相似文献
18.
Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma 总被引:9,自引:0,他引:9
In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation. 相似文献
19.
V Ventrella F Trombetti A Pagliarani G Trigari A R Borgatti 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,95(1):95-105
1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship. 相似文献
20.
Two kinds of ATP binding sites were found to exist on the ATPase molecule. One was the catalytic site (1 mol/mol phosphorylation site) and its apparent dissociation constant for ATP was about 1 microM. The other was the regulatory site(s) and its apparent dissociation constant for ATP was equal to or higher than about 0.2 mM. The affinities of both sites for AMPPNP were three times lower than those for ATP. The affinity of the ATPase for ATP was reduced by the addition of KCl, but unaffected by the addition of NaCl. As thermodynamically expected, the affinity of the Na+-binding sites for Na+ ions was almost completely unaffected by the addition of ATP, which markedly decreased that of the K+-binding sites for K+ and Rb+ ions. In the absence of KCl, Na+ ions were bound very rapidly to the Na+-binding sites [(1979) J. Biochem. 86, 509--523]. However, Na+ ions were bound very slowly to the enzyme preincubated with 50 microM KCl, and the Na+ binding was markedly accelerated by the addition of ATP or AMPPNP at concentrations much higher than several microM. On the other hand, in the presence of 50 microM KCl, 1 mol of ATP was bound to the catalytic site with the same dissociation constant as that in the absence of KCl, and another 1 mol of ATP bound with a dissociation constant of about 0.1 mM. Therefore, we concluded that the Na+ binding to the enzyme in a K+ form is markedly accelerated by the binding at ATP to the regulatory site. 相似文献