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1.
When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.  相似文献   

2.
Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.  相似文献   

3.
The requirement of extracellular Ca2+ for insulin action has been indicated by past studies. With a view to understand the interaction of insulin with Ca2+ in the vicinity of the cell membrane, we have examined the ability of insulin and its constituent polypeptide chains A and B to translocate Ca2+ and Mg2+ across the lipid bilayer in two sets of synthetic liposomes. The first were unilamellar vesicles made of dimyristoylphosphatidylcholine and contained the Ca2+ sensor dye arsenazo III. Peptide-mediated Ca2+ and Mg2+ transport in these vesicles was monitored at 37 degrees C in a neutral buffer containing CaCl2 or MgCl2 using a difference absorbance method. In the second set, multilamellar vesicles of egg lecithin containing trapped fura-2 were employed and the cation transport was followed at 20 degrees C by fluorescence changes in the dye. Control experiments indicated that the hormonal peptides caused no appreciable perturbation of the vesicles leading to leakage of contents or membrane fusion. In both liposome systems, substantial Ca2+ and Mg2+ transport was observed with insulin and the B chain; the A chain was less effective as an ionophore. Quantitative analysis of the transport kinetic data on the B chain showed a 1:1 peptide-Ca2+ complex formed inside the membrane. In light of the available structural data on Ca2+ binding by insulin and insulin receptor, our results suggest the possibility of the hormone interacting with the receptor with the bound Ca2+.  相似文献   

4.
Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.  相似文献   

5.
It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.  相似文献   

6.
The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.  相似文献   

7.
The effects of adrenaline on 45Ca2+-exchange curves for isolated hepatocytes incubated under various steady-state conditions were investigated. Kinetic analysis showed that the simplest compartment configuration consistent with each set of data was a series configuration of a three-compartment closed system comprising compartment 1 (C1), the extracellular medium, and two kinetically distinct compartments of cellular exchangeable Ca2+, C2 and C3 (C1 = C2 = C3). Subcellular fractionation of hepatocytes labelled with 45Ca2+ at 0.1 mM-Ca2+ indicated that C3 includes exchangeable Ca2+ in the mitochondria and endoplasmic reticulum. The following results were obtained from experiments conducted at 37 degrees C at five different extracellular Ca2+ concentrations. For both untreated and adrenaline-treated cells, plots of the flux from C1 to C2 as a function of the extracellular Ca2+ concentration were best described by straight lines consistent with Ca2+ influx across the plasma membrane being a diffusion process. Adrenaline increased the value of the permeability constant for Ca2+ influx by 40%. For untreated cells, plots of the flux between C2 and C3 as a function of the concentrations of Ca2+ in these compartments approached a plateau at high Ca2+ concentrations. Adrenaline caused a 3-fold increase in the concentration of Ca2+ that gives half-maximal rate of Ca2+ transport from C2 to C3. At 1.3 mM extracellular Ca2+, a decrease in incubation temperature from 37 degrees C to 20 degrees C decreased the quantity of Ca2+ in C3 and the flux and fractional transfer rates for the transport of Ca2+ between C2 and C3. At 20 degrees C adrenaline increased the quantity of Ca2+ in C3 and the fractional transfer rates for the transfer of Ca2+ from C1 to C2, and from C2 to C3. At 37 degrees C and 2.4 mM extracellular Ca2+, antimycin A plus oligomycin decreased the quantity of Ca2+ in C3 and increased the fractional transfer rate for the transport of Ca2+ from C3 to C2. In the presence of antimycin A and oligomycin, adrenaline did not increase the quantity of Ca2+ in C2 or the flux and fractional transfer rate for the transport of Ca2+ from C1 to C2, whereas these parameters were increased in the absence of the inhibitors.  相似文献   

8.
Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.  相似文献   

9.
The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.  相似文献   

10.
Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium   总被引:1,自引:0,他引:1  
Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.  相似文献   

11.
Structural and functional properties of a Ca2+-ATPase from human platelets   总被引:3,自引:0,他引:3  
An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.  相似文献   

12.
Cadmium (Cd) uptake and secretion across the apical membrane of epithelial cells was studied using LLC-PK1 cells cultured on Petri dishes and permeable membranes, respectively. Cd accumulation in cells from the apical medium was decreased by low temperature and metabolic inhibitors. A saturable tendency was observed between initial Cd accumulation and increased concentrations of Cd in the apical medium at 37 degrees C, but not at 4 degrees C. Co-incubation with ZnCl2 or CuCl2 competitively decreased Cd accumulation at 37 degrees C. A decrease in the pH of the apical medium markedly decreased Cd accumulation. Pretreatment of cells with an inorganic anion-exchange inhibitor significantly decreased Cd uptake at pH 7.4 in the presence of bicarbonate, but only marginally in its absence. A decrease in the pH of the apical medium increased the secretory (basolateral-to-apical) transport of Cd, with a concomitant decrease in the cellular accumulation of Cd. Co-incubation with Cd and tetraethylammonium, a typical substrate of the organic cation transporter, decreased Cd transport, with a concomitant increase in cellular Cd accumulation. The uptake and secretion of Cd across the apical membrane appear to be partly mediated via an inorganic anion exchanger and a H+ antiport of the organic cation transport system, respectively. Therefore, a decrease in pH of the apical medium markedly decreases Cd accumulation, possibly as a result of not only the decrease in Cd uptake via an inorganic anion exchanger, but also the increase in Cd secretion via the Cd2+/H+ antiport. Further evidence of the antiport was obtained from experiments using brush border membrane vesicles isolated from rat kidney and small intestine. In addition, passive diffusion of Cd appears to be decreased by low temperature and a decrease in pH.  相似文献   

13.
Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.  相似文献   

14.
The interactions of Cd2+ with active Ca2+ transport systems in rat intestinal epithelial cells have been investigated. ATP-driven Ca2+ transport in basolateral plasma membrane vesicles was inhibited by Cd2+ with an I50 value of 1.6 nM free Cd2+ at 1 microM free Ca2+, using EGTA and HEEDTA to buffer Ca2+ and Cd2+ concentrations, respectively. The inhibition was competitive in nature since the Km value of Ca2+ increased with increasing Cd2+ concentrations while the Vmax remained constant. Cd2+ had similar effects on ATP-dependent Ca2+ uptake by permeabilized enterocytes, indicating that non-mitochondrial and mitochondrial Ca2+ stores are also inhibited by nanomolar concentrations of Cd2+. We conclude that ATP-driven Ca2+ transport systems are the most sensitive elements so far reported in Cd2+ intoxication.  相似文献   

15.
Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.  相似文献   

16.
Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).  相似文献   

17.
ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain   总被引:1,自引:0,他引:1  
Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.  相似文献   

18.
The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.  相似文献   

19.
We have identified and characterized calcium transport and the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in plasma membrane vesicles prepared from rat liver. The calcium transport did not absolutely require the presence of oxalate and was completely inhibited by 1 microM of ionophore A23187. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle and liver microsomes, was not absolutely required to maintain the net accumulation of calcium. The Vmax and Km for calcium uptake were 35.2 +/- 10.1 pmol of calcium/mg of protein/min, and 17.6 +/- 2.5 nM of free calcium, respectively. Ten mM magnesium was required for the maximal accumulation of calcium. Substitution of 5 and 10 mM ADP, CTP, GTP, and UTP for ATP could not support calcium uptake. The calcium uptake was not affected by 0.5 mM ouabain, 20 mM azide, or 2 micrograms/ml of oligomycin but was inhibited in a dose-dependent fashion by vanadate, with a Ki of approximately 20 microM for vanadate. The substrate affinities and specificities of this calcium-transport activity suggest that it is closely associated with the (Ca2+ + Mg2+)-ATPase reported in the plasma membranes of liver (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). A calcium-stimulated and magnesium-dependent phosphoprotein was also demonstrated in the same membrane vesicles. The free calcium concentration at which its phosphorylation was half-maximal was 15.5 +/- 5.6 nM. Sodium fluoride, ouabain, sodium azide, oligomycin, adriamycin, and N,N'-dicyclohexylcarbodiimide did not affect its formation while vanadate at 100 microM inhibited the calcium-dependent phosphorylation by approximately 60%. The properties of this phosphoprotein suggest that it may be the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in the plasma membranes of rat liver.  相似文献   

20.
A cyclohexanemonocarboxylic acid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for Pb2+ and Cd2+, transporting them across a phospholipid bilayer membrane. Transport studies were carried out using 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) vesicles containing the chelating indicator 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2). Data obtained at pH 7.0 using this system, show that the synthetic ionophore transports divalent cations with the selectivity sequence Pb2+ > Cd2+ > Zn2+ > Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. Selectivity factors, based on the ratio of individual initial cation transport rates, are 280 (Pb2+/Ca2+), 62 (Pb2+/Zn2+), 68 (Cd2+/Ca2+), and 16 (Cd2+/Zn2+). Plots of log initial rate versus logM(n+) or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 cation-ionophore complex, but that complexes with other stoichiometries may also be present. The ionophore transports Pb2+ and Cd2+ by a predominantly electrogenic mechanism, based upon an enhanced rate of transport that is produced by agents which dissipate transmembrane potentials. The rate of Pb2+ transport shows a biphasic pH dependence with the maximum occurring at pH approximately 6.5. The high selectivity for Pb2+ and Cd2+ displayed by the cyclohexanecarboxylic acid-capped 15-crown-5 ether suggests potential applications of this ionophore for the treatment of Pb and Cd intoxication, and removal of these heavy metals from wastewater.  相似文献   

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