共查询到20条相似文献,搜索用时 15 毫秒
1.
Regulation of Ca2+ transport in brain mitochondria. I. The mechanism of spermine enhancement of Ca2+ uptake and retention 总被引:3,自引:0,他引:3
Spermine enhances electrogenic Ca2+ uptake and inhibits Na(+)-independent Ca2+ efflux in rat brain mitochondria. As a result, Ca2+ retention by brain mitochondria increases greatly and the external free Ca2+ level at steady-state can be lowered to physiologically relevant concentrations. The stimulation of Ca2+ uptake by spermine is more pronounced at low concentrations of Ca2+, effectively lowering the apparent Km for Ca2+ uptake from 3 microM to 1.5 microM. However, the apparent Vmax is also increased. At low Ca2+ concentrations, Ca2+ uptake is diffusion-limited. Spermine strongly inhibits Ca2+ binding to anionic phospholipids and it is suggested that this increases the rate of surface diffusion which reduces the apparent Km for uptake. The same effect could inhibit the Na(+)-independent efflux if the rate of efflux is limited by Ca2+ dissociation from the efflux carrier. In brain mitochondria (but not in liver) the spermine effect depends on the presence of ADP. In a medium that contains physiological concentrations of Pi, Mg+, K+, ADP and spermine, brain mitochondria sequester Ca2+ down to 0.1 microM and below, depending on the matrix Ca2+ load. Moreover, brain mitochondria under the same conditions buffer the external medium at 0.4 microM, a concentration at which the set point becomes independent of the matrix Ca2+ content. Thus, mitochondria appear to be capable of modulating calcium oscillations in brain cells. 相似文献
2.
Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol. 相似文献
3.
Interactions of physiological ligands with the Ca pump and Na/Ca exchange in squid axons 总被引:3,自引:2,他引:1
下载免费PDF全文
![点击此处可从《The Journal of general physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have studied the interaction of physiological ligands other than Nai and Cai with the Ca pump and Na/Ca exchange in internally dialyzed squid axons. The results show the following. (a) Internal Mg2+ is an inhibitor of the Nao-dependent Ca efflux. At physiological Mg2+i (4 mM), the inhibition amounts to approximately 50%. The inhibition is partial and noncompetitive with Cai, and is not affected by Nai or ATP. The ATP-dependent uncoupled efflux is unaffected by Mgi up to 20 mM. Both components of the Ca efflux require Mg2+i for their activation by ATP. (b) At constant membrane potential, Ki is an important cofactor for the uncoupled Ca efflux. (c) Orthophosphate (Pi) activates the Nao-dependent Ca efflux without affecting the uncoupled component. Activation by Pi occurs only in the presence of Mg-ATP or hydrolyzable ATP analogues. Pi under physiological conditions has no effect on the uncoupled component; nevertheless, at alkaline pH, it inhibits the Ca pump, probably by product inhibition. (d) ADP is a potent inhibitor of the uncoupled Ca efflux. The Nao-dependent component is inhibited by ADP only at much higher ADP concentrations. These results indicate that (a) depending on the concentration of Ca2+i, Na+i Mg2+i, and Pi, the Na/Ca carrier can operate under a low- or high-rate regime; (b) the interactions of Mg2+i, Pi, Na+i, and ATP with the carrier are not interdependent; (c) the effect of Pi on the carrier-mediated Ca efflux resembles the stimulation of the Nao-dependent Ca efflux by internal vanadate; (d) the ligand effects on the uncoupled Ca efflux are of the type seen in the Ca pump in red cells and the sarcoplasmic reticulum. 相似文献
4.
Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells 总被引:2,自引:0,他引:2
B E Corkey J T Deeney M C Glennon F M Matschinsky M Prentki 《The Journal of biological chemistry》1988,263(9):4247-4253
Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed. 相似文献
5.
Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant. 相似文献
6.
L de Meis 《The Journal of biological chemistry》1991,266(9):5736-5742
The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled). 相似文献
7.
E J Harris 《The Biochemical journal》1979,178(3):673-680
The efflux of Ca2+ from rat heart mitochondria has been examined by using Ruthenium Red to inhibit active uptake after predetermined loadings with Ca2+. The efflux is proportional to the internal Ca2+ load; it is increased by Na+ applied when the mitochondria are respiring and this effect is inhibited by oligomycin. The efflux of Ca2+ is diminished by ATP and by ADP, with the latter the more effective. Both active uptake and efflux of Ca2+ are slowed by bongkrekic acid; this action has a time lag. The lower efflux found with the nucleotides and with bongkrekic acid seems to correspond to the more condensed state seen in the electron microscope when these agents are applied [Stoner & Sirak (1973) J. Cell Biol. 56, 51-64, 65-73]. The results are discussed in relation to the less-permeable state being contingent upon nucleotide binding to the membrane. 相似文献
8.
The reversal of the rat liver microsomal Ca2+-ATPase transport cycle was studied. Microsomes were loaded with 45Ca2+ (approximately 30 nmol/mg of protein) in an ATP-dependent process, and the time dependency of the microsomal 45Ca2+ efflux was determined with various ADP and inorganic phosphate (Pi) concentrations. Pseudo-first-order rate constants (K'e) for 45Ca2+ efflux were determined. Although there was considerable 45Ca2+ efflux in the absence of added ADP or Pi, the addition of ADP or Pi alone had minimal effects upon the K'e; in contrast, a 2.5-fold increase in the K'e was observed in the presence of both ADP and Pi. The apparent Km values for ADP and Pi were 4 microM and 0.22 mM, respectively. Stimulation of 45Ca2+ efflux by ADP and Pi was associated with ATP synthesis. The calcium ionophore A23187 prevented ATP synthesis, which indicates that the Ca2+ gradient facilitates the coupling of ATP synthesis to Ca2+ efflux. 相似文献
9.
The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane. 相似文献
10.
Y H Lau 《Biochimica et biophysica acta》1983,730(2):276-284
Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange. 相似文献
11.
The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane. 相似文献
12.
Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In this study we investigated fluctuations in mitochondrial membrane potential (DeltaPsim) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K+-based buffer containing 20 microM EDTA, supplemented with malate and glutamate, and rhodamine 123 for DeltaPsim determination. DeltaPsim fluctuations were triggered by mitochondrial Ca2+ uptake since they were inhibited by both ruthenium red, a Ca2+-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca2+ (approximately 30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited DeltaPsim fluctuations, with maximal effects occurring at 100 microM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K+-channels are not involved in the mechanism of DeltaPsim fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of DeltaPsim. We suggest DeltaPsim fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca2+ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane. 相似文献
13.
The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria. 总被引:3,自引:0,他引:3
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed. 相似文献
14.
ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist. 相似文献
15.
L de Meis 《The Journal of biological chemistry》1976,251(7):2055-2062
The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis. 相似文献
16.
The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange. 相似文献
17.
Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis. 相似文献
18.
The kinetics of influx and efflux of 45Ca and its accumulation by the subcellular membranes of adipose tissue have been studied. The initial rate of Ca2+ efflux does not depend on the intracellular concentration of Na+ and K+. The rate of exchange between intracellular 45Ca and 40Ca of the incubation medium is independent on concentration of Na+ and K+ in the incubation mixture. This suggests the absence of Na,Ca-transmembrane exchange in the adipocytes. The changes in the ratio of intracellular concentration of Na+ and K+ by the factors inhibiting the activity ofNa,K-ATPase cause redistribution of Ca in the intracellular pools of the adipocytes. The lypolytic agents (adrenalin, adrenocorticotropic hormone, caffeine) but not dibutytyl-3' : 5'-AMP, accelerate Ca2+ efflux from the adipocytes. At physiological concentrations of ATP, succinate and Pi the highest Ca-accumulating activity is observed in adipose tissue mitochondria. The highest initial rate of Ca uptake, as in the case of contractile tissues, is detected in the endoplasmic reticulum membranes. In contrast to the plasma membranes and reticulum, in which the Ca-accumulating capacity is independent of ATP concentration up to 0.5 mM, the Ca-accumulating capacity of mitochondria decreases 8--9-fold with a reduction in ATP concentration from 4 down to 1 mM. The physiological significance of this phenomenon in the action mechanism of lipolytic agents, which reduce the ATP content in the adipocytes, is discussed. 相似文献
19.
The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane. 相似文献
20.
The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway. 相似文献