Contribution of the translocator of adenine nucleotides and the ATP synthase to the control of oxidative phosphorylation and arsenylation in liver mitochondria

The regulation of the rate of mitochondrial oxidative phosphorylation and arsenylation was studied at two external free Ca2+ concentrations. The rate of arsenate-stimulated respiration in absence of added ADP was not affected by external 10(-9) and 10(-6) M Ca2+ levels or carboxyatractyloside, while state 3 respiration was profoundly modified. In addition, the kinetic analysis showed that the rate of arsenylation in the presence of ADP was more efficient (Vm/Km ratio 3.5 times higher) in the catalytic process than phosphorylation. Therefore, this suggests that the activity of the ATP/ADP carrier is importantly controlled by Ca2+. The evaluation of the control in phosphorylation showed that the flux-control coefficients (Ci) exerted by the ATP/ADP carrier (ranged between 0.23 and 0.48) and the ATP synthase (0.05-0.57) were modified in a reciprocal way by Ca2+ and Pi concentrations. This suggests that these two enzymes are coupling sequentially through a common intermediate, the intramitochondrial ATP/ADP ratio. Other important steps controlling phosphorylation were the b-c1 complex (Ci = 0.30) and the cytochrome oxidase (Ci = 0.23) but they were not modified by Ca2+. It was also found that the main step controlling arsenylation was the ATP synthase (Ci = 0.74). The increment in the inorganic arsenate concentration induced a diminution in the control exerted by the ATP synthase (from 0.73 to 0.56). The results suggest that Ca2+ and Pi (or inorganic arsenate) could be regulated by ATP synthesis through an activating effect on ATP/ADP carrier and/or ATP synthase.     

Regulation of oxidative phosphorylation in the inner membrane of rat liver mitochondria by calcium ions

The effect of accumulation of Ca2+ at physiological concentrations (10(-8)-10(-6) M) on the rates of ATP synthesis and hydrolysis in rat liver mitochondria was studied. An addition of 5 x 10(-7) M Ca2+ resulted in the maximal rates of synthesis and hydrolysis of ATP. Decrease in the concentration of Ca2+ to 10-8 M or its increase to 5 x 10(-6) M inhibited oxidative phosphorylation and ATP hydrolysis. It was found that the rate of oxidative phosphorylation correlated with the phosphorylation level of a 3.5-kD peptide in the mitochondrial inner membrane on varying the Ca2+ concentration. The possible regulation of oxidative phosphorylation in mitochondria by Ca2+ is discussed.     

Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes

Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

The intensity of oxidative phosphorylation and the function of the adenylate system in the liver mitochondria of active and hibernating susliks Citellus undulatus

The state of adenylate system and intensity of oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels were studied depending on the concentration of extramitochondrial Ca2+ ([Ca2+]ex). It was shown that at [Ca2+]ex.10(-7) M, the content of ATP as well as ATP/ADP ratio are slightly lower in the mitochondria of hibernating ground squirrels than in the mitochondria of active animals. The other parameters of the adenylate system under the same conditions differ insignificantly. [Ca2+]ex increase to 10(-6) M has little effect on the parameters of the adenylate system of active animals. On the contrary, the mitochondria of hibernating ground squirrels are strongly affected: the level of ATP is 1.5-fold and the ratio of ATP/ADP is almost 2-fold decreased. At both [Ca2+]ex the intensity of oxidative phosphorylation is essentially higher in the mitochondria of active ground squirrels. With increasing [Ca2+]ex the rate of ATP synthesis decreases, and in the mitochondria of hibernating animals the decrease is more pronounced than in the mitochondria of active animals. Thus, oxidative phosphorylation and adenylate system of mitochondria from hibernating ground squirrels are more sensitive to [Ca2+]ex increase than those of the mitochondria of active animals.     

Effects of photodynamic action on energy coupling of Ca2+ uptake in liver mitochondria

In mitochondria isolated from rat liver, incubated in the presence of 6 X 10(-3) mM hematoporphyrin and irradiated with UV light at 365 nm, respiration, oxidative phosphorylation and Ca2+ uptake were measured in order to determine the respective photosensitivity of these functions. Irradiation with increasing doses produces uncoupling of oxidative phosphorylation followed by inhibition of Ca2+ uptake and finally arrest of respiration. Ca2+ uptake stimulated by the addition of ATP was also studied in mitochondria uncoupled by irradiation which were still able to concentrate Ca2+ aerobically. Anaerobic Ca2+ uptake driven by ATP hydrolysis was found to be similar in control and in irradiated mitochondria, suggesting a different photosensitivity for the ATPase as compared to the ATP-synthase activity.     

Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.     

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.     

Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.     

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Mg2,Ca2+-ATPase activity of sarcolemmas of intestinal smooth muscle cells in the rabbit

Preparations of rabbit small intestine smooth muscle cell sarcolemma are capable of hydrolyzing ATP in the presence of millimolar concentrations of Mg2+ and Ca2+ and possess the activity of Mg2+,Ca2+-ATPase having a high affinity for Ca2+ (Km = 5.8 X 10(-6) M). The optimal conditions for the Mg2+,Ca2+-ATPase reaction were established. It was demonstrated that sarcolemmal preparations hydrolyze ATP, GTP, ITP and UTP almost at the same rates. The enzyme contains SH-groups that are unequally exposed to the water phase and are inhibited by 50% by p-chloromercurybenzoate and by 90% by dithionitrobenzoate. The Mg2+,Ca2+-ATPase activity is highly sensitive to oxytocin: at the concentration of 10(-7) MU/ml, the hormone completely inhibits the enzyme without affecting its Mg2+-, Ca2+- and Na+,K+-ATPase activities.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.     

Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

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.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.