Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Regulation of the monovalent cation permeability of brain mitochondria

The Na+ and K+ conductances of rat brain mitochondria were estimated from rates of metabolically dependent swelling and uncoupling of respiration. These were maximal in the presence of EDTA plus Pi. Pi could not be replaced with acetate. Na+ conductance was greater than that of K+ and was therefore examined in greater detail. According to the influences of N-ethylmaleimide, internal Pi (exogenous and perhaps endogenous) promoted Na+ permeability. Treatment with the ionophore A23187 obviated the Pi requirement although EDTA was still necessary. The stimulation by EDTA with Pi or A23187 and inhibition by exogenous Mg2+ suggested endogenous polyvalent cations could also regulate Na+ conductance. The influence of these substances upon endogenous Mg2+ (and Ca2+) levels is consistent with such a role of membrane-bound Mg2+. Low levels of ruthenium red (150 pmol/mg) inhibit Na+ permeation, indicating that the number of 'sites' or 'channels' involved may be small. The Ca2+ uniport is not directly involved in Na+ flow according to its greater sensitivity to inhibition by ruthenium red.     

Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Respiratory control and ADP:O coupling ratios of isolated chick heart mitochondria

Heart mitochondria isolated from 14- to 21-day-old chicks are highly coupled and often have respiratory control ratio (RCR) values exceeding 100. This paper presents data from a study of some of the properties of these mitochondria. The studies show that: (a) The ADP:O ratios and the state 4 rates of respiration are highly dependent upon the concentration of mitochondria at which these parameters are measured. (b) The mitochondrial isolate is contaminated with at least two divalent cation-stimulated ATPase, of which one is the F1F0-ATPase of broken mitochondria. (c) The oligomycin-sensitive component of state 4 respiration is completely inhibited by ethylene glycol bis(beta-amino-ethylether) N,N'-tetraacetic acid (EGTA). This inhibition is biphasic and attributable to the differential affinity of EGTA for Ca(II) and Mg(II). (d) Ca(II) and Mg(II) stimulate state 4 respiration, thereby depressing RCR values. These cations also decrease ADP:O ratios from greater than or equal to 3.25 to 3.0 for some NAD-linked substrates. (e) Uncoupled (i.e., oligomycin-insensitive) state 4 respiration can be abolished by treating the mitochondria with Nagarse and by preincubating mitochondria with exogenous substrate. (f) The ADP:O ratios obtained when these heart mitochondria oxidize pyruvate/malate, alpha-ketoglutarate, and beta-hydroxybutyrate are fractional and significantly greater than 3.0.     

The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria.

The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed.     

Kinetics of nucleotide transport in rat heart mitochondria studied by a rapid filtration technique

A rapid filtration technique has been used to measure at room temperature the kinetics of ADP and ATP transport in rat heart mitochondria in the millisecond time range. Transport was stopped by cessation of the nucleotide supply, without the use of a transport inhibitor, thus avoiding any quenching delay. The mitochondria were preincubated for 30 s either in isotonic KCl containing succinate, MgCl2, and Pi (medium P) or in isotonic KCl supplemented only with EDTA and Tris (medium K); they were referred to as energized and resting mitochondria, respectively. The kinetics of [14C]ADP transport in energized mitochondria were apparently monophasic. The plateau value for [14C]ADP uptake reached 4-5 nmol of nucleotide.(mg of protein)-1. Vmax values for [14C]ADP transport of 400-450 nmol exchanged.min-1.(mg of protein)-1 with Km values of the order of 13-15 microM were calculated, consistent with rates of phosphorylation in the presence of succinate of 320-400 nmol of ATP formed.min-1.(mg of protein)-1. The rate of transport of [14C]ATP in energized mitochondria was 5-10 times lower than that of [14C]ADP. Upon uncoupling, the rate of [14C]ATP uptake was enhanced, and that of [14C]ADP uptake was decreased. However, the two rates did not equalize, indicating that transport was not exclusively electrogenic. Transport of [14C]ADP and [14C]ATP by resting mitochondria followed biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Ca2+ transport in brain mitochondria. II. The mechanism of the adenine nucleotides enhancement of Ca2+ uptake and retention

ADP greatly enhances the rate of Ca2+ uptake and retention in Ca2+ loaded mitochondria. Atractyloside, a specific inhibitor of the ADP/ATP translocator, completely inhibits the ADP effect, while bongkrekate, another specific inhibitor of the translocator enhances the effect of ADP. These results indicate that locking the ADP/ATP translocator in the M-state is sufficient to produce the ADP effect. Cyclosporin A, a specific inhibitor of the Ca2(+)-induced membrane permeabilization does not substitute for ADP, indicating that ADP directly affect the rate of electrogenic Ca2+ uptake. The effect of the translocator conformation on the rate of electrogenic Ca2+ uptake is independent of the concentration of Pi and is not caused by changes in membrane potential. However, locking the carrier in the M-state appears to increase the negative surface charge on the matrix face of the inner membrane. This may lead to an enhanced rate of Ca2+ dissociation from the electrogenic carrier at the matrix surface. The rate of Na(+)-independent Ca2+ efflux is only slightly inhibited by locking the carrier in the M-state, presumably due to the same mechanism. In the presence of ADP, Pi inhibits the Na(+)-independent efflux. In the presence of physiological concentrations of spermine, Pi and Mg2+, the rate of Ca2+ uptake, Ca2+ retention and Ca2+ set points depend sharply on ADP concentration at the physiological range of ADP. Thus, changes of cytosolic ADP concentration may lead to change in the rate of Ca2+ uptake by mitochondria and thus modulate the excitation-relaxation cycles of cytoplasmic free calcium.     

Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate, Mg(2+), and other effectors

The interplay of inorganic phosphate (Pi) with other ligands such as Mg(2+), ADP, ATP, and Ca(2+) on the activation of 2-oxoglutarate dehydrogenase complex (2-OGDH) in both isolated enzyme complex and mitochondrial extracts was examined. Pi alone activated the enzyme, following biphasic kinetics with high (K(0.5) = 1.96+/-0.42 mM) and low (K(0.5) = 9.8+/-0.4 mM) affinity components for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values below 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-OGDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelating the free Mg(2+) and also as a Mg-ATP complex. With or without Mg(2+), ADP, and Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V(m) of the reaction; ATP diminished the V(m), but it increased the affinity for 2-OG in the mitochondrial extract. Pi did not modify the 2-OGDH activation by Ca(2+). The results above mentioned were similar for both preparations, except for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of this study indicated that physiological concentrations of Pi may exert a significant activation of 2-OGDH, which was potentiated by Mg(2+) and high pH, but surpassed by ADP.     

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.     

Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), spring (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p less than or equal to 0.05). In the 51 day E group ATPase activity (0.378 +/- 0.009 mumol Pi X mg-1 X min-1) was greater than at 10 and 21 days (0.307 +/- 0.006 and 0.323 +/- 0.008 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). No change occurred in the S group from 10 to 21 and 51 days (p greater than or equal to 0.05). Both the 21 and 51 day S (0.318 +/- 0.011 and 0.399 +/- 0.010 mumol Pi X mg-1 X min-1) and E (0.323 +/- 0.008 and 0.378 +/- 0.009 mumol Pi X mg-1 X min-1) groups had higher activity compared to the C group (0.193 +/- 0.029 and 0.172 +/- 0.031 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). Maturation (10--51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca2+ binding (p less than or equal to 0.05) while Ca2+ uptake ability did not change (p greater than or equal to 0.05). SR yield, Ca2+ binding and uptake were not altered with S training (p greater than or equal to 0.05). The E training resulted in greater Ca2+ uptake at 51 days compared to C and S (p less than or equal to 0.05), with no change in Ca2+ binding (p greater than or equal to 0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.     

Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria

The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases free Mg2+ by 0.12-0.17 mM depending on the substrate. This decrease in free Mg2+ appears to reflect changing ligand availability in the matrix. The decrease is prevented when the Pi transporter is blocked by mersalyl. Addition of ADP to initiate state 3 respiration causes a marked increase in free matrix Mg2+ (0.1-0.2 mM) that persists as long as ATP formation is taking place; free Mg2+ then returns to the base level. This cyclic change is blocked by oligomycin and carboxyatractyloside and appears to reflect to a large extent the decrease in matrix Pi that accompanies oxidative phosphorylation. Exchange of external ADP for matrix ATP may also contribute to the increase in free matrix Mg2+. Addition of an uncoupler promotes anion efflux and increases free matrix Mg2+. Similar changes in free Mg2+ on addition of Pi, ADP, or uncoupler are seen when extramitochondrial Mg2+ is buffered from 0.5 to 2 mM, but the basal free matrix Mg2+ increases as external Mg2+ concentration increases in this range. Free matrix Mg2+ also increases when total mitochondrial Mg2+ is increased by respiration-dependent uptake in the presence of Pi. It is concluded that matrix free Mg2+ changes significantly with changing ligand availability and that such changes may contribute to the regulation of Mg2(+)-sensitive matrix enzymes and membrane transporters.     

Enzymes and processes of activation of the endometrium stromal cells

The endometrium stroma cells and properties of such key enzymes as acetylcholinesterase, Mg2+, Ca(2+)-ATPase, AMP-deaminase have been investigated in them. The activity of acetylcholinesterase in suspension of cells compounds is 9.8 +/- 0.2 mumol of tiocholinbromide/mg protein/hour and is reduced under influence of exogenous ATP, NO2-, H2O2 and Triton X-100. Common Mg2+, Ca(2+)-ATPase activity of compounds of 36 +/- 2 mumol Pi/mg protein/hour, is depressed by sodium azide and thapsigargine, that specifies presence of an investigated enzyme in mitochondria and endoplasmic reticulum of investigated cells. In a suspension of stroma cells with addition of 0.2% of Triton X-100 for augmentation of permeability of cellular membranes and 1.5 M KCl for a dissociation of complexes AMP-deaminase with proteins and membranes, the deamination exogenous AMP up to IMP and NH3, is registered generated in the given response. The supposition about NH3 role as the paracrine regulator in the system endometrium-myometrium of the uterus is expressed.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria

Ruthenium red-insensitive, uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria is much slower than from rat liver mitochondria under comparable conditions. In the presence of Pi and at moderate or high Ca2+ loads, ruthenium red-insensitive Ca2+ efflux elicited with uncoupler is approximately 20 times more rapid for rat liver than Ehrlich cell mitochondria. This is attributed to resistance of tumor mitochondria to damage by Ca2+ due to a high level of endogenous Mg2+ that also attenuates Ca2+ efflux. Calcium release from rat liver and tumor mitochondria is inhibited by exogenous Mg2+. This applies to ruthenium red-insensitive spontaneous Ca2+ efflux associated with Ca2+ uptake and uncoupling, and (b) ruthenium red-insensitive Ca2+ release stimulated by uncoupling agent. The endogenous Mg2+ level of Ehrlich tumor mitochondria is approximately three times that of rat liver mitochondria. Endogenous Ca2+ is also much greater (six fold) in Ehrlich tumor mitochondria compared to rat liver. Despite the quantitative difference in endogenous Mg2+, the properties of internal Mg2+ are much the same for rat liver and Ehrlich cell mitochondria. Ehrlich ascites tumor mitochondria exhibit slow, metabolically dependent Mg2+ release and rapid limited release of Mg2+ during Ca2+ uptake. Both have been observed with rat liver and other types of mitochondria. The proportions of apparently "bound" and "free" Mg2+ (inferred from release by the ionophore, A23187) do not differ significantly between tumor and liver mitochondria. Thus, the endogenous Mg2+ of tumor mitochondria has no unusual features but is simply elevated substantially. Ruthenium red-insensitive Ca2+ efflux, when expressed as a function of the intramitochondrial Ca2+/Mg2+ ratio, is quite similar for tumor and rat liver. It is proposed, therefore, that endogenous Mg2+ is a major regulatory factor responsible for differences in the sensitivity to damage by Ca2+ and Ca2+ release by Ehrlich ascites tumor mitochondria compared to mitochondria from normal tissues.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Effect of caffeine on azide-sensitive Mg2+, ATP-dependent increase of tetracycline fluorescent response in modeling of Ca ions accumulation in mitochondria

The influence of caffeine on the Mg2+, ATP-dependent Ca(2+)-uptake was investigated in the experiments, conducted on mitochondria isolated from myometrium of nonpregnant estrogenized rats. NaN3-sensitive CTC fluorescence increasing (lambda f = = 520 nm) was used as a test for active Ca2+ transport. Kinetics of NaN3-sensitive Mg2+, ATP-dependent component of CTC fluorescence change fits to the pattern of the first-order reaction either in the absence or in the presence of caffeine (20 mM). Caffeine (0-20 mM) inhibited both the stationary level (settled on the 2-3d min. of incubation) and the initial rate V0, and rate constant k of CTC fluorescence change. Magnitude of the apparent inhibition constant I0.5 for caffeine is 10.41 +/- 1.81 mM, inhibition process has weak positive cooperativity--the value of apparent Hill coefficient for caffeine is equal to 1.2 +/- 0.3. Data obtained suggest that caffeine inhibits both stationary Ca2+ capacity of mitochondria and the rate of NaN3-sensitive Mg2+, ATP-dependent Ca(2+)-accumulation in case of myometrium. These data could be useful for further investigation of molecular and membrane mechanisms of caffeine action on the intracellular Ca2+ homeostasis in uterus smooth muscle and its contractive activity.     

Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates

Microsomal fractions, highly enriched with endoplasmic reticulum of rat and human liver exhibit Ca2+ uptake catalyzed by a Ca2+-pumping ATPase. The mechanism of Ca2+-translocation involves: (i) reversible Ca2+-dependent formation of an acyl-phosphoenzyme intermediate (Mr 116,000 to 118,000) with bound Ca2+, which in the reversed reaction can transphosphorylate its Pi to ADP to re-synthesize ATP; (ii) reversible transition of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, not further reactive to ADP; (iii) hydrolytic cleavage, stimulated by Mg2+, K+, and ATP of the ADP-unreactive phosphoenzyme with liberation of Pi. By analogy to a mechanism proposed for the Ca2+ pump of sarcoplasmic reticulum, the translocation of Ca2+ to and dissociation from the inner side of the membrane is suggested to occur by a conformational change, coupled with a decrease in Ca2+-affinity of the phosphoenzyme during its transition into the ADP-unreactive isomer. With CaATP as the effective substrate the reactions proceed normally but at a considerably slower rate.     

Effects of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate.

1. The reduction of mitochondrial NAD(P) by 2-oxoglutarate was monitored as a measure of 2-oxoglutarate dehydrogenase activity in its intramitochondrial locale. In the absence of ADP, steady-state reduction of NAD(P) by 0.5 mM-2-oxoglutarate in the presence of 0.5 mM-L-malate was markedly increased by extramitochondrial Ca2+, with 50% activation at pCa 6.58, when the Na+ concentration was 10 mM, the Pi concentration ws 5 mM and the added Mg2+ concentration was 1 mM. Omission of Pi resulted in 50% activation at pCa 6.77; omission of Mg2+ resulted in 50% activation at pCA greater than or equal to 7.3. 2. The activation of 2-oxoglutarate dehydrogenase could be reversed on addition of an excess of EGTA. The rate of inactivation was dependent on the concentration of Na+, with K0.5 2.5 mM, which is consistent with the rate of withdrawal of Ca2+ from the mitochondria being the limiting factor. 3. The steady-state reduction of cytochrome c by 2-oxoglutarate (0.5 mM) also showed a marked dependence on pCa in the absence of ADP; in the presence of an excess of ADP, no such effect of Ca2+ was detectable. 4. Mitochondria from the hearts of senescent rats showed an undiminished rate of dehydrogenase activation by Ca2+ but a rate of inactivation by excess EGTA that was diminished by 40%. Direct studies of Ca2+ egress with Arsenazo III confirmed a decrement in rate with old age. 5. Studies of 2-oxoglutarate dehydrogenase activity as a function of the mitochondrial context of Ca2+, as measured by atomic-absorption spectrophotometry, showed half-maximal activation at a mitochondrial content of 1.0 nmol of Ca2+/mg of protein, and saturation at 3 nmol/mg. 6. These findings support the model advanced by Denton, Richards & Chin [(1978) Biochem. J. 176, 899-906], of a control of the tricarboxylate cycle by intramitochondrial Ca2+, and demonstrate the range of mitochondrial Ca2+ content over which this may occur. In addition, they raise the possibility of a disturbance of this control mechanism in old age.