Rickettsial permeability. An ADP-ATP transport system.

The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP.     

Adenine nucleotide and lysine transport in Chlamydia psittaci.

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Characterization of pyrophosphate exchange by the reconstituted adenine nucleotide translocator from mitochondria

The transport of inorganic pyrophosphate (PPi) by the adenine nucleotide translocator from beef heart mitochondria was studied in a reconstituted system. The transport of PPi is dependent on appropriate transmembrane substrates. The activity of PPi exchange is about one tenth as compared to the ADP/ATP exchange, whereas the transport affinity for PPi is very low (2-5 mM). The adenine nucleotide carrier catalyzes a strict counterexchange of PPi and nucleotides with an exchange stoichiometry close to 1. The inhibitor specificity of PPi exchange is comparable to that of ADP/ATP exchange.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

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.     

Studies on adenine nucleotide exchange in mitochondria from the muscle tissue of Ascaris lumbricoides (Nematoda)

Adenine nucleotide exchange between the intra- and extramitochondrial compartments of mitochondria isolated from the muscle tissue of Ascaris lumbricoides was investigated. The exchange was specific for ATP and ADP, AMP, adenosine and non-adenine nucleotides were not exchanged at significant rates. All combinations of counter exchange were found to be possible between intra- and extramitochondrial ATP and ADP. Adenine nucleotide exchange in Ascaris muscle mitochondria was inhibited by atractyloside; was strongly temperature dependent; activated by potassium and magnesium and only slightly activated by calcium. The K_m for adenine nucleotide exchange in Ascaris mitochondria was 4·1 and 2·85 μm for ATP and ADP respectively. The properties of adenine nucleotide exchange in Ascaris muscle mitochondria are thus similar in general features to the adenine nucleotide translocase system of mammalian mitochondria.     

Increased adenine nucleotides in liver mitochondria after mannoheptulose injection in vivo

In adult rats, mannoheptulose injection causes a transient decrease in the serum insulin-to-glucagon ratio and a concomitant increase in serum glucose concentration. These effects attain a maximum 1 h after the injection and then decline toward normal. Correlated with the hormone changes is a dramatic increase in the adenine nucleotide content (ATP + ADP + AMP) of liver mitochondria, which peaks to over 50% of control values at 1 h. The increase in mitochondrial adenine nucleotides must occur by uptake from the cytosol, because the adenine nucleotide content of the whole tissue remains constant. The accumulation of adenine nucleotides by the mitochondria probably occurs over the recently characterized carboxyatractyloside-insensitive transport pathway that allows exchange of ATP-Mg for Pi. The actual mechanism by which net uptake is regulated after mannoheptulose injection has not yet been elucidated; however, changes in the Km or Vmax of the carrier and an increase in the tissue ATP/ADP ratio were eliminated as possibilities. The increase in matrix adenine nucleotide content in response to hormone changes brought about by mannoheptulose was much greater and more reproducible than what is achieved with glucagon injection. Mannoheptulose treatment may therefore be preferable as a model for further study of hormone effects on mitochondrial function.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria.

1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

The relation of sodium and potassium ion transport to the respiration and adenine nucleotide content of liver slices treated with inhibitors of respiration

1. The dependence of the net transport of Na(+) and K(+) by rat liver on the respiration has been determined by incubating slices in the presence of varying concentrations of respiratory inhibitors. 2. Neither the rate of net transport nor the total amount of each ion transported was inhibited unless the rate of endogenous respiration was decreased below a critical value of about 330mmol of O(2)/h per kg of protein (i.e. 50% of the total endogenous respiration). 3. The uninhibited rate of respiration could be varied over a twofold range (380-770mmol of O(2)/h per kg of protein) by the use of different substrates, but the critical value for the onset of transport inhibition was quite constant (290-360mmol/h per kg of protein) under these different conditions. 4. Slices incubated at 38 degrees C without inhibitors showed an increase of their ATP content and the concentration ratio ATP/ADP. The final ATP content and concentration ratio, ATP/ADP, of slices treated with different concentrations of inhibitors were closely related to the rate of respiration. 5. The increased ATP content of the control slices during incubation was equal to the increase of total adenine nucleotides. At increasing degrees of respiratory inhibition the relative contributions of ADP and AMP to the total adenine nucleotide content increased. 6. The critical rate of respiration for the onset of inhibition of ion transport and the corresponding contents of adenine nucleotides provide estimates of the maximal values of certain parameters of energy metabolism required for the support of alkali-cation transport in the liver slices.     

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.     

Energy Metabolism of Rickettsia typhi: Pools of Adenine Nucleotides and Energy Charge in the Presence and Absence of Glutamate

The obligate intracellular bacterium Rickettsia typhi was examined for its ability to generate and maintain an adenylate energy charge in an extracellular environment. Freshly purified organisms were incubated, at 34 degrees C and pH 7.4, with or without glutamate and various other metabolites, and the levels of ATP, ADP, and AMP were determined. Of the metabolites tested, glutamate and glutamine were the most effective for the generation of ATP. In the presence of glutamate, there was a rapid increase in the level of ATP, followed by a moderate decrease during 150 min of incubation. The energy charge increased from a level of 0.2 to 0.5 to about 0.7 to 0.75, and then slowly declined to about 0.45 to 0.6. In the absence of glutamate, after an occasional initial surge in ATP level as the temperature was changed from 4 to 34 degrees C, there was a sharp decline in both ATP and energy charge (to 0.1 and sometimes to 0.01). The rickettsiae maintained their ability to regenerate their energy charge upon the addition of glutamate for about 30 min, but this ability declined with further incubation. In contrast to Escherichia coli, the decline in ATP in R. typhi was accompanied by a sharp increase in the level of AMP and the total adenylate pool. No adenine or adenosine was recovered from rickettsiae incubated with labeled AMP, ADP, or ATP. From these experiments and the demonstration reported elsewhere that rickettsiae transport the adenine nucleotides, it can be concluded that the adenylate energy charge in R. typhi is governed by the salvage of the adenine nucleotides rather than their unphosphorylated precursors. Thus, R. typhi undergoes greater shifts in energy charge than other bacteria, a phenomenon which may account for their instability in an extracellular environment. Under optimal conditions the adenylate energy charge of R. typhi approaches levels that border on those generally regarded as adequate for growth.     

Uric acid synthesis by rat liver supernatants from purine bases,nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g^?1 of wet liver min^?1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo-succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min^?1 per mg^?1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM ) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.