Changes in tissue levels of carnitine and other metabolites during myocardial ischemia and anoxia

The induction of ischemia in the open chest dog, or anoxia in the perfused rat heart, causes dramatic changes in the tissue levels of free acyl carnitine and related metabolites. During the early phase of ischemia or anoxia the tissue levels of free carnitine decline, while acetyl carnitine rapidly increases. These changes are accompanied by elevation in long-chain acyl carnitine, long-chain acyl CoA, and lactate and by decreases in acetyl CoA, CoA, ATP, and creatine phosphate. As the degree of ischemia becomes more severe, carnitine appears to be lost from the myocardium. A scheme is presented which relates carnitine-linked mitochondrial metabolism to the activity of carnitine acyl transferase, ANT, carnitine/acyl carnitine translocase, creatine phosphokinase, and pyruvate dehydrogenase. It is suggested that the conversion of carnitine to acyl carnitine during the onset of ischemia may play an important role, by virtue of its effect on these enzymes, in the regulation of metabolism during the early or reversible phase of ischemia.     

Metabolic changes induced by maximal exercise in human subjects following L-carnitine administration

In double-blind cross-over experiments, ten moderately trained male subjects were submitted to two bouts of maximal cycle ergometer exercise separated by a 3 day interval. Each subject was randomly given either L-carnitine (2 g) or placebo orally 1 h before the beginning of each exercise session. At rest L-carnitine supplementation resulted in an increase of plasma-free carnitine without a change in acid-soluble carnitine esters. Treatment with L-carnitine induced a significant post-exercise decrease of plasma lactate and pyruvate and a concurrent increase of acetylcarnitine. The determination of the individual carnitine esters in urine collected for 24 h after the placebo exercise trial revealed a decrease of acetyl carnitine and a parallel increase of a C4 carnitine ester, probably isobutyrylcarnitine. Conversely, acetylcarnitine was strongly increased and C4 compounds were almost suppressed in the L-carnitine loading trial. These results suggest that L-carnitine administration prior to high-intensity exercise stimulates pyruvate dehydrogenase activity, thus diverting pyruvate from lactate to acetylcarnitine formation.     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

Oxidation of acetate,acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O₂ uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.     

The properties and regulation of pantothenate kinase from rat heart

Pantothenate kinase (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33), the first enzyme in the pathway of CoA synthesis, was partially purified from rat heart. A study of the properties of the kinase showed that it possesses a broad pH optimum between 6 and 9, is activated or inhibited nonspecifically by various anions, and has MgATP as the nucleotide substrate. The Km for MgATP is 0.6 mM and that for pantothenate is 18 microM. CoA and acyl esters of CoA are inhibitors of the kinase with the inhibition by acetyl-CoA being only slightly greater than that by free CoA. The inhibition by free CoA is uncompetitive with respect to pantothenate concentration, with a Ki for inhibition of 0.2 microM. L-Carnitine was found to be a nonessential activator of the kinase. This compound had no effect by itself but specifically reversed the inhibition of the kinase by CoA. The Ka for deinhibition by L-carnitine is 0.27 mM. Free carnitine content was measured in perfused hearts and is found to vary in correlation with perfusion conditions that are known to alter rates of intracellular phosphorylation of pantothenate. These properties of pantothenate kinase provide a potential mechanism for the control of CoA synthesis. The enzyme is regulated by feedback inhibition by CoA and its acyl esters and this inhibition is modified by changes in the concentration of free carnitine.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

Trypanosoma brucei brucei: the catabolism of glycolytic intermediates by digitonin-permeabilized bloodstream trypomastigotes and some aspects of regulation of anaerobic glycolysis

1. The production of pyruvate, glycerol and glycerol-3-phosphate by intact and digitonin-permeabilized Trypanosoma brucei brucei has been studied with glucose or the glycolytic intermediates as substrates. 2. Under aerobic conditions hexosephosphates gave maximal glycolysis in the presence of 40-60 micrograms digitonin/10(8) trypanosomes while the triosephosphates gave it at 20-30 micrograms digitonin/10(8) trypanosomes. 3. In the presence of salicylhydroxamic acid, and the glycolytic intermediates, permeabilized trypanosomes produced equimolar amounts of pyruvate and glycerol-3-phosphate and no glycerol. Under the same conditions, glucose catabolism produced glycerol in addition to pyruvated and glycerol-3-phosphate. 4. In the presence of salicylhydroxamic acid and ATP or ADP intact trypanosomes produced equimolar amounts of pyruvate and (glycerol plus glycerol-3-phosphate) with glucose as substrate. 5. A carrier for ATP and ADP at the glycosomal membrane is implicated. 6. It is apparent that glycerol formation is regulated by the ATP/ADP ratio and that it needs intact glycosomal membrane and the presence of glucose.     

Regulation of purified rat liver acetyl CoA carboxylase by phosphorylation

Acetyl CoA carboxylase was purified from liver of fasted-refed rats to near homogeneity, based on electrophoretic analysis and biotin content. These preparations contained an endogenous protein kinase that catalyzed the transfer of radioactive phosphate from [gamma-32P]ATP to acetyl CoA carboxylase, accompanied by a decrease in acetyl CoA carboxylase activity. Phosphate incorporated into acetyl CoA carboxylase was removed when the preparation was incubated with partially purified phosphorylase phosphatase catalytic subunit with regain of enzymatic activity. This endogenous protein kinase was shown not to be affected by either cyclic-AMP-dependent protein kinase inhibitor, EGTA, or trifluoperazine. The addition of either cyclic-AMP or purified cyclic-AMP-dependent protein kinase catalytic subunit to the purified acetyl CoA carboxylase preparation increased protein phosphorylation but had no further effect on acetyl CoA carboxylase activity. Purified acetyl CoA carboxylase was shown to act as an ATPase during the phosphorylation reaction.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

Pathways of acetate and propionate production in adult Fasciola hepatica

In adult F. hepatica pyruvate is decarboxylated via pyruvate dehydrogenase to acetyl-CoA; acetyl-CoA is then cleaved to acetate via three possible mechanisms (1) carnitine dependent hydrolysis, (2) CoA transferase, (3) reversal of a GTP dependent acyl-CoA synthetase. Of these three systems, CoA transferase has by far the greatest activity. Propionate production by F. hepatica is similar to the mammalian system, succinate being metabolized via succinic thiokinase, methylmalonyl-CoA isomerase, methyl-malonyl-CoA racemase and propionyl-CoA carboxylase to propionyl-CoA. Propionyl-CoA is then cleaved to propionate by the same three pathways as acetyl-CoA. No ATP or GTP production could be demonstrated when acetyl- or propionyl-CoA were incubated with homogenates of F. hepatica. This indicates that carnitine dependent hydrolysis or CoA transferase are the major pathways of acetyl- or propionyl-CoA breakdown. The CoA transferase reaction would result in the conservation of the bond energy although there is no net ATP synthesis.     

Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei

Oxidative phosphorylation and substrate level phosphorylation catalyzed by succinyl-CoA synthetase found in the citric acid and the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle contribute to mitochondrial ATP synthesis in procyclic Trypanosoma brucei. The latter pathway is specific for trypanosome but also found in hydrogenosomes. In organello ATP production was studied in wild-type and in RNA interference cell lines ablated for key enzymes of each of the three pathways. The following results were obtained: 1) ATP production in the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle was directly demonstrated. 2) Succinate dehydrogenase appears to be the only entry point for electrons of mitochondrial substrates into the respiratory chain; however, its activity could be ablated without causing a growth phenotype. 3) Growth of procyclic T. brucei was not affected by the absence of either a functional citric acid or the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle. However, interruption of both pathways in the same cell line resulted in a growth arrest. In summary, these results show that oxygen-independent substrate level phosphorylation either linked to the citric acid cycle or tied into acetate production is essential for growth of procyclic T. brucei, a situation that may reflect an adaptation to the partially hypoxic conditions in the insect host.     

Effect of ischemia on fatty acid metabolism in fetal lung

The effects of ischemia on in vivo fatty acid metabolism in fetal lung were studied using rabbit fetuses of 25 to 28 gestational age. Ischemia was produced by inflating the aortic balloon thereby reducing the uterine blood flow. Ischemic insult resulted significant increase in lactate/pyruvate and NADH/NAD ratios and decrease in ATP/ADP ratio in fetal lung. Levels of CoA, acetyl CoA, carnitine and acetyl carnitine decreased while those of long chain acyl CoA and long chain acyl carnitine enhanced. Tissue content of these metabolites returned to normal after 2 hr stabilization following 20 min of ischemic insult. Ischemia also caused small increase in lipogenesis and neutral lipid content of fetal lungs. Our results thus suggest that β-oxidation in fetal lung is inhibited and becomes rate-limiting for fatty acid oxidation during ischemia.Sudden occurrence of hypoxia or ischemia in the fetus is a typical challenge for the obstetricians. The patients occasionally suffer from neurological injury following cerebral hypoxemia. The hypoxic insult may also affect the respiratory activity significantly. For example, acute alveolar hypoxia causes pulmonary vasoconstriction by damaging pulmonary vascular smooth muscle (1) and results in reduction of fatty acid oxidation by limiting the ATP supply required for metabolic processes (2). Hypoxia has also been shown to decrease the rate of palmitate incorporation into phospholipids (3), inhibit rate of fatty acid synthesis (3) and depress rate of incorporation of fatty acid and phosphatidic acid into lipids (4). Despite the fact that fatty acids represent a major substrate for energy metabolism in lung, no work has been done on the fatty acid metabolism in fetal lung. The present study was designed to determine the fate of fatty acid oxidation in fetal lung during ischemic challenge. The levels of acyl CoA and acylcarnitine intermediates were also measured in order to determine the rate-controlling steps of fatty acid metabolism in the fetal lung.     

Carbon Dioxide Fixation by Extracts of Streptococcus faecalis var. liquefaciens

Extracts of cells of Streptococcus faecalis var. liquefaciens strain 31 incorporated (14)CO(2) into aspartate. Dialyzed extracts produced radioactive oxalacetate in the absence of exogenously added glutamate and pyridoxal-5'-phosphate and produced radioactive aspartate in the presence of these components. Reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate could not be substituted for adenosine triphosphate (ATP); phosphoenolpyruvate even in the presence of nucleoside diphosphates could not replace pyruvate plus ATP; propionate plus coenzyme A (CoA) could not replace pyruvate in supporting CO(2) fixation by cell extracts. Fixation by dialyzed cell extracts required pyruvate, ATP, MgSO(4), and was stimulated by biotin, KCl, 2-mercaptoethanol, CoA, and acetyl CoA. Inhibition of fixation occurred when avidin, NaCl, oxalacetate, or aspartate was added to dialyzed extracts. On the basis of the products formed and the effects of substrates and cofactors on the fixation reaction, it was concluded that pyruvate carboxylase is responsible for CO(2) fixation in this microorganism.     

Effects of acute acid-base changes on rat renal pyruvate dehydrogenase. Renal pyruvate dehydrogenase during acid-base alterations

Glutamine, the principal source of urinary ammonia, can be fully oxidized or converted to glucose by the kidney. To be oxidized, the carbon skeleton of glutamine must enter the TCA cycle as acetyl CoA formed by pyruvate dehydrogenase (PDH). The purpose of this study was to measure kidney PDH activity (active and total) following acute acid-base changes in vivo. PDHa activity was elevated after acute metabolic alkalosis and acidosis and unchanged by respiratory acidosis. Kidney ADP/ATP, CoA/acetyl CoA and calculated mitochondrial NAD+/NADH ratios were also determined and revealed an increase in kidney ADP/ATP with alkalosis but no changes during metabolic and respiratory acidosis.     

ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production. Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase. However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes. To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK). The parasite was not affected by PPDK gene knockout. In contrast, inhibition of PYK expression by RNA interference was lethal for these cells. In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced. Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes. We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.     

Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

2′,3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme.     

Regulation of mammalian pyruvate dehydrogenase

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase. Evidence is presented that all components of the pyruvate dehydrogenase complex including the phosphatase and kinase are located within the inner mitochondrial membrane. Direct measurements of the matrix concentration of substrates and effectors is not possible by techniques presently available. This is the key problem in the identification of the mechansims involved in the alterations in pyruvate dehydrogenase activity observed in adipose tissue and muscle. A number of indirect approaches have been used and these are reviewed. Most hopeful is the recent finding in this laboratory that in both adipose tissue and heart muscle, differences in activity of pyruvate dehydrogenase in the intact tissue persist during preparation and subsequent incubation of mitochondria.     

Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.