Uptake of fatty acids by cultured cardiac cells from chick embryo: evidence for a facilitation process without energy dependence

Cultured heart cells from chick embryo accumulate fatty acids up to 50 fold at a steady-state level under defined conditions [ref.1]. Studies of fatty acid uptake as a function of different cellular parameters (intracellular ATP and pH, membrane potential and electrochemical gradients for monovalent and divalent cations) show the lack of effect of these factors. The rate of uptake is temperature-dependent. The maximum velocity V is affected with no change in the Km value of the saturable component; the activation energies were found to be 35.5 kJ.mol-1 for palmitate and 42 kJ.mol-1 for oleate. The results are in favour of a facilitation process which leads to an accumulation of fatty acids without energy dependence. The accumulation of fatty acids could be due to their association to intracellular membraneous and/or cytosolic components.     

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.     

Recombination kinetics following nanosecond laser photolysis of carbonmonoxyhaemogloblin

The kinetics of the ultrafast ligand recombination following 347 nm laser photolysis of aqueous solutions of carbonmonoxyhaemogloblin have been investigated. The process is biphasic and the rate constants for the two processes as functions of temperature have been used to give activation energies of 6 +/- 3.9 kJ . mol-1 for the fast process and 31 +/- 4.8 kJ . mol-1 for the slow process. Frequency factors have also been calculated. The two processes are discussed in relation to both low-temperature studies and model calculations on the rate of entry of carbon monoxide into haem proteins.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

A ligand-induced, temperature-dependent conformational Change in penicillopepsin. Evidence from nonlinear Arrhenius plots and from circular dichroism studies

The effect of temperature on the rate constants of hydrolysis of various substrates by penicillopepsin is dependent on the length of the substrate. For the series Ac-(Ala)m-Lys-Nph-(Ala)n-amide (where Ac- is acetyl- and Nph- is p-nitrophenylalanyl-), where m and n = 0-2, substrates lacking both P'2 and P3 residues give linear Arrhenius plots with an energy of activation of about 55 kJ.mol-1. The Arrhenius plots of substrates in which an alanine residue occupies P'2 show a sharp break at an average transition temperature of 10.5 degrees C. The activation energies are approximately 90 kJ.mol-1 below and approximately 54 kJ.mol-1 above the transition temperature, respectively. For substrates in which P3 is occupied, the average transition temperature is 14.2 degrees C. In this case, the activation energies are 66 kJ.mol-1 below and from 26 to 39 kJ.mol-1 above the transition point. The most probable explanation of these phenomena is that substrate interaction at subsites S3 and/or S'2 of the enzyme induces a temperature-dependent conformational change. Physical evidence for this comes from the observation that the temperature dependence of a CD absorption band at 242 nm of a penicillopepsin-pepstatin complex shows a sharp break that corresponds to those observed in the Arrhenius plots of substrates with alanine at P'2 and P3, whereas the same CD band in the free enzyme is linearly dependent on temperature.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. I. Anion antiport in Vero cells

In sodium-free buffer of low ionic strength, the uptake of chloride and sulfate in Vero cells was found to occur mainly by antiport which was very sensitive to inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Efflux of anions from the cells appeared to energize the uptake. While the uptake of Cl- occurred over a wide pH range, that of SO4(2-) showed a clear maximum at pH 6-7. The rate of efflux of 36Cl- and 35SO4(2-) was strongly increased by the presence of permeant anions in the efflux buffer. Preincubation of the cells at slightly alkaline pH strongly increased the rate of C1- efflux into buffers nominally free of permeant anions, as well as the efflux by exchange. This increase did not occur if the cells were depleted for ATP during the preincubation. Depolarization of the cells reduced the rate of efflux into buffers without permeant anions, indicating that the efflux is at least partly due to net, electrogenic, anion transport. The efflux by antiport was not affected by manipulations of the membrane potential, indicating electroneutral exchange. The uptake and efflux were increased to the same extent with increasing temperature, the activation energies were Ea = 25 kcal/mol of Cl- and Ea = 12 kcal/mol of SO4(2-). Similar anion antiport appears to occur in L, baby hamster kidney, and HeLa S3 cells.     

The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to pKa values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined. Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.     

Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n- propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.     

Calcium binding to calmodulin and its globular domains

The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.     

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.     

Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility v. At temperatures above 303 K, this time-dependent change in v is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pKa of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pKa. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ X mol-1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.     

Comparison of the membrane transport of anthracycline derivatives in drug-resistant and drug-sensitive K562 cells.

One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells. This is correlated with the presence in the membrane of resistant cells of a 150-180-kDa glycoprotein, P-glycoprotein, responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4'-o-tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe. The data show that the uptake occurs through free permeation of the neutral form of the drug. For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+(7.2) = V+/[D]e.n where V+ and [D]e are the initial rate of uptake and the concentration of drug in the medium at pHe = 7.2 respectively and n is the number of cells. This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer. Efflux coefficients k-(7.2)- at pHi = 7.2 (the intracellular pH value) have also been calculated. In the case of sensitive cells, k+(7.2) and k-(7.2)- are equal. For resistant cells, the efflux coefficient is composed of two terms: (a) (k-)p corresponding to the passive diffusion of the neutral form of the drug and (k-)p = k+; (b) (k-)a corresponding to an active efflux mediated by the P-glycoprotein. Our data suggest that the anthracycline derivatives efflux actively in the neutral form.     

Kinetic and thermodynamic parameters for tRNA binding to the ribosome and for the translocation reaction.

Kinetic analyses of tRNA binding to the ribosome and of the translocation reaction showed the following results. 1) The activation energy for the P site binding of AcPhe-tRNA to poly(U)-programmed ribosomes is relatively high (Ea = 72 kJ mol-1; 15 mM Mg2+). If only the P site is occupied with deacylated tRNA(Phe), then the E site can be filled more easily with tRNA(Phe) (no activation energy measurable) than the A site with AcPhe-tRNA (Ea = 47 kJ mol-1; 15 mM Mg2+). 2) A ribosome with blocked P and E sites represents a standard state of the elongation cycle, in contrast to a ribosome with only a filled P site. The two states differ in that AcPhe-tRNA binding to the A site of a ribosome with prefilled P and E sites requires much higher activation energy (87 versus 47 kJ mol-1). The latter reaction simulates the allosteric transition from the post- to the pretranslocational state, whereby the tRNA(Phe) is released from the E site upon occupation of the A site (Rheinberger, H.-J., and Nierhaus, K. H. (1986) J. Biol. Chem. 261, 9133-9139). The reversed transition from the pre- to the posttranslocational state (translocation reaction) requires about the same activation energy (90 kJ mol-1). 3) Both elongation factors EF-Tu and EF-G drastically reduce the respective activation energies. 4) The rate of the A site occupation is slower than the rate of translocation in the presence of the respective elongation factors. The data suggest that the A site occupation rather than, as generally assumed, the translocation reaction is the rate-limiting step of the elongation cycle.     

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.     

Relationship between lipid fluidity and water permeability of bovine tracheal epithelial cell apical membranes

Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.     

Ornithine/phosphate antiport in rat kidney mitochondria. Some characteristics of the process

[14C]Ornithine uptake by rat kidney mitochondria has been investigated according to the stop inhibitor method by using praseodimium chloride as an inhibitor. The existence of an ornithine/Pi exchange was found occurring with 1:1 stoichiometry. Both uptake and efflux follow first-order kinetics with a k of 2.4 min-1. Uptake increases with increasing pH. The activation energy for the process is 58.6 kJ/mol and Q10 is 2.6. Ornithine/Pi exchange is electrical and energy-dependent, as suggested by the sensitivity of the process to the ionophores valinomycin and nigericin. Measurements both of proton movement across the mitochondrial membrane and of membrane potential strongly suggest that ornithine uptake into mitochondria is driven by the electrochemical proton gradient via the dependent ornithine/Pi translocator and delta pH-dependent Pi carrier.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum.

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.