Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

Role of intracellular buffering power on the mitochondria-cytosol pH gradient in the rat liver perfused at 4 degrees C

The factors regulating the amplitude and the pH gradient between cytosol and mitochondria (DeltapHmito-cyt) were investigated in the isolated rat liver perfused at 4 degrees C. Liver ATP content, pH, and buffering power of cytosolic and mitochondrial compartments were evaluated in situ using phosphorus-31 nuclear magnetic resonance spectroscopy. No DeltapHmito-cyt was detected in the liver perfused without bicarbonate. Permeant weak acid in the perfusate (H2CO3, 25 mM, or isobutyric acid, 25, 50, or 100 mM) acidified both cytosol and mitochondria and revealed a DeltapHmito-cyt from 0.06 to 0.31 pH unit. Nevertheless, the manipulations of the DeltapHmito-cyt were more effective under bicarbonate-free conditions, due to the absence of buffering by H2CO3/HCO-3. In the absence of bicarbonate, the intracellular buffering power was threefold higher in the mitochondria (110 mmol/pH unit at pHmito 7.16) than in the cytosol (44 mmol/pH unit at pHcyt 7.30) and dependent on the matrix and cytosol pH, respectively. These buffering powers were almost double in the presence of bicarbonate. In the bicarbonate-free perfused liver, the respiratory activity was 0.08 +/- 0.02 micromol O2/min. g liver wet weight and the ATP turnover was only 40 +/- 7 nmol/min. g liver wet weight, indicating the weak activity of liver mitochondria when DeltapHmito-cyt was <0.05 pH unit. The ATP turnover during a 50 mM isobutyric acid load was 35 +/- 4 nmol/min. g liver wet weight whereas DeltapHmito-cyt rose to 0.26 +/- 0.02 pH unit and pHmito remained alkaline. Hence, although DeltapHmito-cyt was increased the ATP turnover remained unchanged. This work is the first evaluation of the mitochondrial buffering power in the isolated liver. The DeltapHmito-cyt observed within various acid loads reflected the differential titration of cytosol and mitochondria containing proteins and H2CO3/HCO-3 buffering systems. Moreover, no direct relationship between DeltapHmito-cyt and ATP turnover could be shown.     

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.     

Proliferation, macromolecular synthesis and energy metabolism of in vitro grown Ehrlich ascites tumor cells after inhibition of ATP-ADP translocation by atractyloside

In the presence of 3 mM atractyloside, growth of in vitro cultured Ehrlich ascites tumor cells is inhibited by 70% within 24 h. Viability of the cells is not severely affected (dye exclusion test). Incorporation of 2-[14C]-thymidine and U-[14C]-leucine into acid insoluble precipitate were reduced by 80% or 20% respectively as compared to controls. Flow cytometric analysis of cell cycle progression revealed a retardation rather than an arrest of cell growth by atractyloside. Morphological changes of the cells primarily concern mitochondria which are spherical shaped with translucent matrix rid of cristae. After transfer of atractyloside treated cells to normal medium, proliferation and macromolecular synthesis normalized within 3 to 6 h. At 3 mM of atractyloside, glucose consumption of the cells increased by 25%, lactate production by 30%. Lactate/glucose ratio was 1.9 after 24 h. Oxygen uptake was reduced by 35% after 12 h. The [ATP]/[ADP] ratio of the whole cells runs through a maximum between 12 and 18 h. The ratio never falls below 5.0. The ATP/ADP concentration ratio in the mitochondrial and extramitochondrial compartment were increased as compared to controls. delta G of ATP hydrolysis of the intact cells was in a normal range (-50 kJ), energy charge was 0.86 (controls 0.88). Transport of amino acids, uptake of glucose and activity of Na+, K+-ATPase of the plasma membrane were not impaired by 3 mM atractyloside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catecholamine and vasopressin stimulation of gluconeogenesis from dihydroxyacetone in the presence of atractyloside

Atractyloside inhibited gluconeogenesis from dihydroxyacetone in hepatocytes from fasted rats and increased lactate synthesis. In the presence of atractyloside, lactate/pyruvate and beta-hydroxybutyrate/aceto-acetate ratios were increased and the accumulation of Fru-2,6-P2 was prevented. In the absence of atractyloside, gluconeogenesis from dihydroxyacetone was stimulated by dibutyryl-cAMP and, to a much lesser extent, by norepinephrine and vasopressin. Omission of Ca2+ increased the stimulation by norepinephrine but prevented that by vasopressin. High concentrations (greater than or equal to 40 microM) of atractyloside abolished the stimulation of gluconeogenesis by dibutyryl-cAMP but not that by norepinephrine or vasopressin. Exogenous Ca2+ was not required for hormonal stimulation in the presence of atractyloside. The stimulation by norepinephrine was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N-tetraacetic acid or prazosin but not by propranolol. Atractyloside caused decreases of all glycolytic intermediates and an activation of pyruvate kinase. Norepinephrine partially reversed these effects. The mitochondrial and cytosolic ATP/ADP ratios were determined by digitonin fractionation of hepatocytes. Norepinephrine or vasopressin increased the cytosolic ATP/ADP in the presence of atractyloside. We suggest that the increased availability of cytosolic ATP could be responsible for the stimulation of gluconeogenesis by these hormones.     

Gluconeogenesis from fructose predominates in periportal regions of the liver lobule

Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected. Rates of glucose and lactate production from 4 mM fructose were around 100 and 75 mumol/g/h, respectively. Rates of fructose uptake were around 190 mumol/g/h when 4 mM fructose was infused. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, decreased glucose formation from fructose maximally by 20% suggesting that a fraction of the lactate formed from fructose is used for glucose synthesis. A good correlation (r = 0.92) between extra oxygen consumed and glucose produced from fructose was observed. At low fructose concentrations (less than 0.5 mM), the extra oxygen uptake was much greater than could be accounted for by glucose synthesis possibly reflecting fructose 1-phosphate accumulation. Furthermore, fructose diminished ATP/ADP ratios from about 4.0 to 2.0 in periportal and pericentral regions of the liver lobule indicating that the initial phosphorylation of fructose via fructokinase occurs in both regions of the liver lobule. Basal rates of oxygen uptake measured with miniature oxygen electrodes were 2- to 3-fold higher in periportal than in pericentral regions of the liver lobule during perfusions in the anterograde direction. Infusion of fructose increased oxygen uptake by 65 mumol/g/h in periportal areas but had no effect in pericentral regions of the liver lobule indicating higher local rates of gluconeogenesis in hepatocytes located around the portal vein. When perfusion was in the retrograde direction, however, glucose was synthesized nearly exclusively from fructose in upstream, pericentral regions. Thus, gluconeogenesis from fructose is confined to oxygen-rich upstream regions of the liver lobule in the perfused liver.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.     

The internal pH and membrane potential of the insulin-secretory granule

1. Incubation of isolated hepatocytes with fructose at concentrations above 3 mM resulted in an apparent inhibition of pyruvate kinase assayed in crude extracts at sub-optimal phosphoenolpyruvate concentrations. 2. Fructose at concentrations below 3 mM caused an activation of the enzyme. 3. Increases in the hepatocyte contents of the positive effectors fructose 1.6-bisphosphate and fructose 1-phosphate were found at all concentrations of fructose up to 10mM. 4. Removal of the extrahepatocyte medium from the hepatocytes by washing resulted in an activation of the enzyme at all concentrations of fructose examined. 5. Inhibitors of the enzyme were shown to accumulate in the hepatocytes despite the depletion of ATP (a known negative effector) caused by higher concentrations of fructose. Indeed the inhibition of pyruvate kinase appeared to be correlated to the depletion of ATP. 6. Alanine (a known inhibitor) was shown to accumulate in hepatocytes as a consequence of incubation with fructose. 7. Allantoin and uric acid were shown to be inhibitors of a partially purified pyruvate kinase preparation assayed both in the presence and in the absence of fructose 1.6-bisphosphate. 8. Allantoin, but not uric acid, accumulated in the extrahepatocyte medium as a result of incubation of the cells with 10 mM-fructose.     

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+.     

Is the adenine nucleotide translocator rate-limiting for oxidative phosphorylation?

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40μm) on O₂ uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K⁺ concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]×[P_i] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K⁺]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett. 74, 50–54] assume. This point is discussed.     

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.     

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

A new approach of the estimation of Km of carbamyl phosphate synthetase for ammonia in isolated rat hepatocytes

1. The Km for ammonia of carbamyl phosphate synthetase was determined by preincubating isolated liver cells for 30 min in the absence of ammonia and bicarbonate and in the presence of ornithine, chloroquine, which blocks lysosomal proteolysis, and aminoxy acetic acid, which inhibits transaminases. 2. The reaction was started with the addition of varying concentrations of ammonia and 10 mM bicarbonate. 3. The rate of citrulline formation was measured as related to ammonia concentration. 4. The pre-incubation with ornithine permits an accumulation of intracellular and mitochondrial ornithine concentrations which in turn allow rapid citrulline formation in the carbamyl phosphate form. 5. This prevents any feedback inhibition on a carbamyl phosphate synthetase or decreases in activity due to accumulation of carbamyl phosphate and/or absence of ornithine. 6. Using these methods in combination with [14C]bicarbonate permitted an estimation of exogenous ammonia for carbamyl phosphate synthesis. 7. The Km for ammonia was 1.5 mM, using a pK of 8.88 the Km for free NH3 was 48 microM.     

Plasmodium falciparum: ATP/ADP transport across the parasitophorous vacuolar and plasma membranes

Previous studies have shown that ATP is required for the growth of the intracellular parasite, Plasmodium, outside its host cell, the erythrocyte, and that bongkrekic acid, an inhibitor of mitochondrial ATP/ADP transporter, inhibits intraerythrocytic Plasmodium maturation. We have characterized ATP/ADP transport of Plasmodium falciparum, isolated by either immune lysis or N2-cavitation. [3H]ATP uptake was due to ATP/ADP exchange since ADP efflux was dependent on exogenous ATP in an approximate 1:1 stoichiometry and both ATP influx and ADP efflux were equally inhibited by atractyloside (Ki = 100 nM). ATP uptake was not inhibited by the nucleoside transport inhibitor, nitrobenzylthioinosine. Conversely, adenosine and hypoxanthine transport were insensitive to atractyloside. ATP influx was characterized by a Km = 0.14 mM and Vmax = 1.2 nmol ATP/min/10(6) cells. Substrate specificity studies for nucleotide-induced ADP efflux indicated a preference for an adenosine ring and triphosphate, but transport did not require a hydrolyzable phosphate bond. Protein synthesis was measured with free parasites starved of glucose. Addition of 1.0 mM ATP resulted in a 40% recovery of total protein synthetic capacity in a process inhibited by 500 nM atractyloside, suggesting that uptake of erythrocyte-derived ATP by P. falciparum may be essential for maintaining maximal rates of protein synthesis during specific stages of intra-erythrocytic parasite maturation.     

Efficiency of oxidative phosphorylation and energy dissipation by H+ ion recycling in rat-liver mitochondrial metabolizing pyruvate.

A method was developed for the calculation of metabolic fluxes through individual enzymatic reactions of pyruvate metabolism including the citric acid cycle in rat liver mitochondrial incubated at metabolic states between state 4 and state 3. This method is based on the measurement of the specific radioactivities of the products formed from [2-14C]pyruvate. With this procedure the energy balance of mitochondria incubated in the presence of [2-14C]pyruvate, ATP, bicarbonate and phosphate at different ATP/ADP ratios in the medium was calculated. The ATP/ADP ratios were maintained at a steady state with creatine kinase plus creatine as a phosphoryl acceptor. The calculations revealed that by adding increasing concentrations of creatine up to 20 mM the energy dissipated by the mitochondria decreased but showed a local maximum at 13mM creatine. Omission of bicarbonate from the medium led to a shift of this maximum. When energy dissipation was minimal the overall P/O ratio was maximal. The amount of energy dissipated was paralleled by the magnitude of the pH gradient across the inner membrane. From these results it was concluded that the recycling of H+ ions which consists of a passive leakage of H+ ions into the matrix and an active extrusion of these ions out of this compartment, is an important energy dissipating process. The H+ ion recycling is thus one of the processes which give rise to the state 4 respiration in mitochondria.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

ATP-dependent activation of a new form of spinach leaf 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase

A novel form of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that possesses little 2-kinase or bisphosphatase activity as isolated has been partially purified from spinach (Spinacia oleracea L.) leaves. However, the new form can be activated by pretreatment with Mg X ATP at room temperature. After ATP activation, the fructose 2,6-bisphosphatase activity has a Michaelis constant for fructose 2,6-bisphosphate of about 1 mM, and is inhibited by high substrate concentrations (greater than 2 mM) and both end products. The kinase/phosphatase activity ratio of the new form was dependent on pH and varied from 0.3 at pH 7.0 to 5.0 at pH 8.2. In contrast, the previously characterized form of the enzyme (which is isolated in an active form and is unaffected by preincubation with Mg X ATP) had an activity ratio of about 2 that was insensitive to pH over the range tested. The ATP-dependent activation of the new enzyme form was stimulated by fructose 6-phosphate and inhibited by glucose 6-phosphate. These results explain why activation is not observed during assay of this enzyme, and indicate that the activation process may be regulated by metabolites. Collectively, these data provide further evidence for the existence, in spinach leaves, of two molecular forms of the enzyme which exhibit different kinetic properties.