共查询到20条相似文献,搜索用时 156 毫秒
1.
Jun Nakamura 《BBA》1983,723(2):182-190
The effects of ATP on Ca2+ binding in the absence of added Mg2+ to the purified sarcoplasmic reticulum Ca2+-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca2+-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca2+ binding was 4·105 M?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH2]PP has little effect on the Ca2+-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca2+ binding. ADP decreased the level of ATP-induced Ca2+ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca2+ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca2+ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl2; after the release of Ca2+, the phosphoenzyme decayed. These observations suggest that Mg2+, added after the ATP-induced Ca2+-binding process, may replace the Ca2+ on the phosphoenzyme and initiate phosphoenzyme decomposition. 相似文献
2.
Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
3.
(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and Pi are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment. 相似文献
4.
Menno De Metz Jocelyne Enouf Marilyne Lebret Sylviane Lévy-Tolédano 《生物化学与生物物理学报:生物膜》1984,773(2):325-328
Several nucleotide triphosphates (NTPs) were tested as energy source for the Ca2+ uptake by human platelet membrane vesicles. The Ca2+ uptake by these membranes was driven by ATP, GTP, ITP, UTP and CTP. The steady-state level of accumulated Ca2+ was equal with the different NTPs. The highest uptake velocity was found with ATP, but about 40–80% of the velocity with ATP could be accomplished with the other nucleotides. The highest affinity was also found with ATP (Km apparent 15 μM). The liberation of Pi from the various NTPs was measured simultaneously with the Ca2+ uptake. The coupling ratio (moles of Ca2+ taken up/moles of Pi liberated) varied from 0.4 for ATP to 2.3 for UTP and was almost independent of the NTP concentration. The enzyme activity with ATP as substrate is strongly dependent on the Ca2+ concentration in contrast to the activity with GTP, ITP, UTP or CTP. 相似文献
5.
Pretreatment of pigeon erythrocyte membrane vesicles with amino acids, ATP, GTP, Pi and some other simple cell constituents (singly and in combination) causes an increase in ATP-dependent Ca2+-uptake activity of vesicles upon subsequent incubation with 45Ca2+ after removal of the above agents from the 'i' face. Amino acids augment the stimulation by all stimulatory agents and are required for stimulation by Pi. The effects of amino acids, ATP, GTP and Pi all occur at physiological concentrations. Many if not all of the effects of the mixture of amino acids that occur naturally in the cells can be accounted for by the group transported by the 'ASC' transport system of Christensen (Christensen, H.N. (1975) Biological Transport, 2nd edn., W.A. Benjamin, Inc., Reading, MA), but not by any single amino acid at its physiological concentration. The effects of ATP and GTP are not mimicked by their non-hydrolysable beta,gamma-imido analogues not by the corresponding 3',5'-cyclic monophosphates. None of the effects described appears to involve calmodulin. We suggest that amino acid transport plays a role in metabolic regulation through effects on cell [Ca2+]. Analogous effects on cell [Ca2+] may be involved in the action of the many hormones which augment amino acid accumulation by the 'A' amino acid transport system. 相似文献
6.
Joel E. Lavine Eric Cantlay Charles T. Roberts Daniel E. Morse 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(1):76-85
Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ >Co2+ >Mn2+ >Fe2+. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties. 相似文献
7.
Michael Sribney Jerre L. Dove Eileen M. Lyman 《Biochemical and biophysical research communications》1977,79(3):749-755
The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP. 相似文献
8.
Avraham Oplatka Raphael Lamed Andras Muhlrad 《Biochimica et Biophysica Acta (BBA)/General Subjects》1975,385(1):20-27
Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg2+ or Ca2+. Splitting of bound ATP was followed by using [γ-3 2P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the Pi-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction. 相似文献
9.
Byung-Chang Suh Jin H. Son Tong H. Joh Kyong-Tai Kim 《Journal of neurochemistry》1997,68(4):1622-1632
Abstract: We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP-and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4? and UTP4? concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ~100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C. 相似文献
10.
Abstract: The present study indicates that central 5-hydroxytryptamine (5-HT; serotonin) receptors can be modulated in opposite directions by Ca2+ and guanine nucleotides [guanosine triphosphate (GTP), β, γ-imidoguanosine 5′-triphosphate (GppNHp)]. Thus CaCl2 (≥0.5 mm ) inhibited whereas GTP and GppNHp (10 μm ) stimulated the 5-HT-sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (Kd ?1) and the number (Bmax) of [3H]5-HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca2+ (≥0.25 mm ); GTP (≥0.1 mm ) and GppNHp (≥0.3; μm ) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [3H]5-HT binding was counteracted by Ca2+: the addition of this cation (5mm -CaCl2) to the assay mixture resulted in a 40-fold increase in the IC50 for GTP; the IC50 for GppNHp increased five-fold under the same condition. The examination of the respective effects of Ca2+ and of GTP on the specific binding of [3H]5-HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca2+-induced increase and of the GTP-induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid-treated rats because [3H]5-HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca2+-induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[3H]5-HT binding sites to adenylate cyclase in the rat brain. 相似文献
11.
Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed. 相似文献
12.
Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells 总被引:1,自引:1,他引:0
Tai-An Lin Kevin D. Lustig Michael G. Sportiello Gary A. Weisman Grace Y. Sun 《Journal of neurochemistry》1993,60(3):1115-1125
Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis off [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]Pi or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D. 相似文献
13.
O. Bârzu F. Eckstein S. Dancea I. Petrescu C. Tărmure L.D. Ngoc A. Hodârnău H.H. Mantsch 《BBA》1979,547(2):361-369
Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria. 相似文献
14.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km 0.25 μM, Vmax 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献
15.
16.
Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [3H]-noradrenaline ([3H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca2+] ([Ca2+]e) was buffered to ~50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ~50% with EC50 values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log10), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular free [Ca2+] ([Ca2+]i). These elevations were reduced at low [Ca2+]e and under these conditions the EC50 values for peak elevation of [Ca2+]i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC50 determinations). At low [Ca2+]e, depletion of nonmitochondrial intracellular Ca2+ stores with the Ca2+-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca2+]i and a minor release of [3H]NA. At low [Ca2+]e, thapsigargin abolished elevation of [Ca2+]i in response to methacholine and bradykinin and completely inhibited their stimulation of [3H]NA release. It is proposed, therefore, that Ca2+ release from Ins(1,4,5)P3-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [3H]NA release in SH-SY5Y cells. 相似文献
17.
A J Ferro A A Vandenbark M R MacDonald 《Biochemical and biophysical research communications》1981,100(2):523-531
In the coupling of ATP pyrophosphorolysis to Ca2+ transport in beef heart mitochondria, for each molecule of ATP cleaved, one proton is released and one Ca2+ is transported into the interior space. With the use of tritium labelled ATP, it could be shown that ATP is pyrophosphorylyzed into a species equivalent to Pi that moves inward, and a species equivalent to ADP that is extruded into the aqueous space on the exterior of the mitochondrion. The species equivalent to Pi has been identified as a negatively charged form of Pi (PO?) and the species equivalent to ADP as a positively charged form (ADP+). The inward flow of PO? is coupled to the inward flow of Ca2+ in 1:1 stoichiometry—a token that Ca2+ must enter in the form of Ca2+A?, where A? is a monovalent anion. During ATP pyrophosphorolysis protons are released on the I side and taken up on the M side—one proton for each molecule of ATP cleaved. The alkalinization of the matrix space leads to the deposition of Ca3(PO4)2 and to the extrusion of the two species released by this deposition (Pi, K+). Two thirds of the PO? is trapped as Ca3(PO4)2 in the matrix space and one third is extruded into the external space. The extrusion of K+ provides a mechanism by which protons can be continuously brought into the matrix space to sustain a high rate of coupled pyrophosphorolysis of ATP. The coupling pattern for Ca2+ transport driven by ATP pyrophosphorolysis is identical with the corresponding pattern for Ca2+ transport driven by electron transfer. This identity is suggestive that coupling mediated by the Fo-F1 system and coupling mediated by electron transfer complexes are mechanistically indistinguishable. 相似文献
18.
Abstract: The effect of replacement of extracellular Na+ with N-methyl-d -glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 µM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4?, replacement of Na+ with NMG had no effect on the ATP-induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4? was >10 µM. The calculated EC50 value for ATP4? stimulation of the [Ca2+]i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP-induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP4?-promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP-differentiated NG108-15 cells. 相似文献
19.
20.
Deborah Dixon Adrian Corbett Duncan H. Haynes 《Journal of bioenergetics and biomembranes》1982,14(2):87-96
The ability of the Ca2+-Mg2+ ATPase pump of skeletal SR to produce and maintain a Ca2+ gradient was studied as a function of the ATP/ADP/Pi ratio. The internal free Ca2+ concentration [Ca2+]i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca2+]i concentration achieved. The inclusion or the omission of 4×10–4 M Pi or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca2+] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca2+]i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca2+ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca2+]i/[Ca2+]o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca2+]i achieved results in trans-inhibition of the pump by tying up the Ca2+ translocator in the inwardly oriented phosphorylated form. The absence of an effect of Pi can be taken as evidence that the dissociation of Ca2+ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme. 相似文献