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1.
Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond. 总被引:1,自引:1,他引:0
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J G McCormack 《The Biochemical journal》1989,264(1):167-174
1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed. 相似文献
2.
Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria. 总被引:10,自引:8,他引:2
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J G McCormack 《The Biochemical journal》1985,231(3):581-595
The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed. 相似文献
3.
In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated. 相似文献
4.
The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed. 相似文献
5.
Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. 总被引:29,自引:24,他引:5
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1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed. 相似文献
6.
Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration. 总被引:2,自引:2,他引:0
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(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+]m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+]m with the extramitochondrial free Ca2+ ([Ca2+]o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+]m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+]m/[Ca2+]o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+]m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+]m/[Ca2+]o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+]m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+]m, as inferred in earlier work. 相似文献
7.
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
8.
J Nedergaard 《European journal of biochemistry》1984,144(1):159-168
The presence and significance of Na+-induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique. Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of 'basal' release of Ca2+ was seen (0.3 nmol Ca2+ X min-1 X mg-1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria. The addition of Na+ in 'cytosolic' levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+ X min-1 X mg-1. This effect was specific for Na+. The significance of this Na+-induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady-state level (set point) induced by Na+. A five-fold increase in this level, from less than 0.2 microM to more than 1 microM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic-anion buffer as in a sucrose buffer. Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+-controlled level, was regained. It was concluded that liver mitochondria should be considered Na+-sensitive, that the capacity of the Na+-induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+-dependent regulators of extramitochondrial ('cytosolic') Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably alpha 1-adrenergic) and changes in this extramitochondrial ('cytosolic') Ca2+ steady state level. 相似文献
9.
Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C. 总被引:1,自引:0,他引:1
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Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed. 相似文献
10.
The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action 总被引:4,自引:0,他引:4
The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166). 相似文献
11.
The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria.
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The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+. 相似文献
12.
Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin. 总被引:7,自引:7,他引:0
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The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls. 相似文献
13.
Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+. 总被引:7,自引:6,他引:1
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Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again. 相似文献
14.
1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2--1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane. 相似文献
15.
Evidence for the existence of regulatory sites for Ca2+ on the Na+/Ca2+ carrier of cardiac mitochondria. 总被引:4,自引:3,他引:1
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The Na+-induced efflux of Ca2+ catalysed by the Na+/Ca2+ carrier of cardiac mitochondria is strongly inhibited by extramitochondrial Ca2+. The nature of this inhibition was investigated as follows. (a) The apparent association of external Na+ and the Ca2+ analogue Sr2+ with substrate-binding sites (i.e. those sites involved in cation translocation) is promoted markedly by K+. The inhibition of Na+/Ca2+ exchange by external Ca2+ is affected little by K+. (b) There is a competitive relationship between the binding of external Na+ and external Ca2+ to substrate-binding sites, whereas at low concentrations (less than 4 microM) extramitochondrial Ca2+ is a partial non-competitive inhibitor with respect to external Na+. (c) This inhibiton by external Ca2+ is characterized by a maximal decrease of about 70% in the Vmax of Na+/Ca2+ exchange and by cooperative binding of external Ca2+ to sites that are half saturated by 0.7-0.8 microM free Ca2+. The binding of Ca2+ and Sr2+ to substrate-binding sites shows no co-operativity. These criteria suggest that the Na+/Ca2+ carrier may contain regulatory sites that render the carrier sensitive to changes in extramitochondrial [Ca2+] within the physiological range. 相似文献
16.
1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates. 相似文献
17.
Sub-micromolar concentrations of extramitochondrial Ca2+ stimulate the rate of citrulline synthesis by rat liver mitochondria.
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1. In the presence of physiological concentrations of Na+ and Mg2+, the rate of citrulline synthesis by isolated rat liver mitochondria respiring on a range of substrates was stimulated by up to 60% when the extramitochondrial Ca2+ concentration was raised from 130 pM to 770 nM. 2. Our findings suggest that hormonal stimulation of the urea cycle may be mediated by Ca2+. 相似文献
18.
The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria. 总被引:2,自引:2,他引:0
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The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+. 相似文献
19.
Regulation of citric acid cycle by calcium 总被引:2,自引:0,他引:2
B Wan K F LaNoue J Y Cheung R C Scaduto 《The Journal of biological chemistry》1989,264(23):13430-13439
The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase. 相似文献
20.
The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix. 相似文献