Cation-activated nucleotidase in cell envelopes of a marine bacterium

Isolated cell envelopes of a marine bacterium, M.B.3, have been prepared which possess a nonspecific, cation-activated nucleotidase. The cell envelope comprises approximately 35% (dry weight) of the whole cell and contains protein, 60.2%; lipids, 20.7%; hexose, 3.4%; and ribonucleic acid, 4.6%. No deoxyribonucleic acid could be detected in the preparations. The nucleotidase has an essential requirement for Mg(2+); maximum activation at pH 8.0 occurs at a divalent cation concentration of approximately 80 mm. At a Mg(2+) to adenosine 5'-triphosphate (ATP) ratio of 2:1, the enzyme was further stimulated by monovalent cations Na(+), K(+), NH(4) (+), and Li(+). Maximum activity was found at a monovalent ion concentration of approximately 0.3 m. The envelope preparation liberated inorganic orthophosphate (P(i)) from ATP, adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) at similar rates. Thin-layer and ion-exchange chromatography show that when AMP, ADP, and ATP were utilized as substrate, approximately 1, 2, and 3 moles of P(i), respectively, were produced per mole of adenosine. P(i) was also liberated from the 5'-triphosphates of guanosine, uridine, and cytidine. The enzyme preparation did not attack p-nitrophenyl phosphate, beta-glycerophosphate, or inorganic pyrophosphate. Sulfhydryl inhibitors p-chloromercuribenzoate, N-ethyl maleimide, and iodoacetate had little effect upon the nucleotidase activity. Ca(2+) and ethylenediaminetetraacetic acid caused complete inhibition of the system, whereas ouabain had no effect upon the enzyme activity. The concentrations of Na(+) (0.3 m) and Mg(2+) ions (60 to 80 mm) required for maximum ATP-hydrolyzing activity were similar to those concentrations necessary for maintenance of cell integrity and for the prevention of cell lysis.     

The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.     

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.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Activation of steroidogenesis and adenylate cyclase by adenosine in adrenal and Leydig tumor cells.

Steroidogenesis by Y-1 adrenal tumor cells in culture is stimulated by ATP, adenyl-5'-yl imidodiphosphate (App(NH)), adenosine 5'(beta, alpha-methylene)triphosphate (App(CH2)p), ADP, AMP, NAD, FAD, and adenosine but not by adenine or other nucleoside triphosphates. ATP, App(NH)p, App(CH2)p, and adenosine are active in the micromolar range. Like adrenocorticotropic hormone (ACTH), the onset of stimulation is immediate and occurs to the same extent. Also active are 2'- and 5'-deoxyadenosine and 2-chloroadenosine whereas adenine xyloside, L-riboside, or arabinoside have very low activity. Stimulation is accompanied by rounding of the cells. Dipyridamole, an inhibitor of adenosine transport, increased the response to low concentrations of adenosine, suggesting that adenosine acts externally. Stimulation of steroidogenesis by adenosine or phosphorylated adenosine compounds fails to occur in the presence of crystalline adenosine deaminase, and the effect of the enzyme on adenosine, ATP, or NAD stimulation is reversed by the competitive inhibitor erythro-9-[3-(nonane-2-ol)]adenine. This suggests that the enzyme acts specifically on adenosine and a requirement for the conversion of the above compounds to adenosine seems probable. The inhibition of cAMP effects by adenosine deaminase suggests that some of its effects are also mediated by conversion to adenosine. Similar stimulation is seen in I-10 Leydig tumor cells, but an ACTH-resistant mutant of Y-1 cells, called OS-3, is relatively resistant to adenosine. Adenosine and 2-chloroadenosine stimulate adenylate cyclase in membranes from Y-1 and I-10 cells at concentrations slightly greater than are effective for steroidogenesis. Other nucleosides are ineffective. Like the NH2-terminal 24 residues of adrenocorticotropic hormone (1-24 ACTH), the adenosine effect in Y-1 membranes is rapid and is on the Vmax intercept (versus ATP) and not on the Km. In contrast to steroidogenesis, adenosine is only a partial agonist for adenylate cyclase. It effect occurs in the presence of ITP, GTP, or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Theophylline inhibits adenosine-stimulated steroidogenesis. Inhibition of adenylate cyclase occurs in the same concentration range but is of the mixed type.     

Stimulation of maximal intracellular insulin action on glycogen synthase by preincubation of adipocytes with adenosine 5'-triphosphate

Preincubation of rat adipocytes with ATP further stimulated maximal insulin action on glycogen synthase. Half-maximum concentration of ATP was 5 X 10(-5) M. ATP, ADP, adenosine, inosine, and GTP were effective, while beta-gamma-methylene ATP was without effect. ADP and GTP were less potent than ATP, adenosine, or inosine. Inosine was active without insulin but was without effect in the presence of insulin. The mechanism of action of adenosine was clearly different from ATP. While ATP required both Mg2+ and Ca2+ for effectiveness, adenosine required only Ca2+. The effect of ATP, but not of adenosine, was preserved after cells were washed. The adenosine effect was completely blocked by theophylline, but the ATP effect was inhibited only 40%. The ATP effect was thus not due to adenosine generated by ATP breakdown.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

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.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

Human placental adenosine kinase. Kinetic mechanism and inhibition

The kinetic properties of human placental adenosine kinase, purified 3600-fold, were studied. The reaction velocity had an absolute requirement for magnesium and varied with the pH. Maximal activity was observed at pH 6.5 with a Mg2+:ATP ranging from 1:1 to 2:1. High concentrations of Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both adenosine and MgATP2-. The Michaelis constant was 0.4 micro M for adenosine and 75 micro M for MgATP2-. Inhibition by adenosine was observed at concentrations greater than 2.5 micro M. AMP was a competitive inhibitor with respect to adenosine and a noncompetitive inhibitor with respect to ATP. ADP was a noncompetitive inhibitor with respect to adenosine and ATP. Hyperbolic inhibition was observed during noncompetitive inhibition of adenosine kinase by AMP and ADP. Other purine and pyrimidine nucleoside mono-, di-, and triphosphates were poor inhibitors in general. S-Adenosylhomocysteine and 2'-deoxyadenosine inhibited adenosine kinase. The data suggest that (a) MgATP2- is the true substrate of adenosine kinase, and both pH and [Mg2+] may regulate its activity; (b) the kinetic mechanisms of adenosine kinase is Ordered Bi Bi; and (c) adenosine kinase may be regulated by the concentrations of its products, AMP and ADP, but is relatively insensitive to other purine and pyrimidine nucleotides.     

Extracellular ATP stimulates polyphosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells. Involvement of a pertussis toxin-sensitive guanine nucleotide binding protein and feedback inhibition by protein kinase C

ATP stimulated a rapid and dose-dependent formation of inositol polyphosphates in rat glomerular mesangial cells. In parallel there was a 80% increase in 1, 2-diacylglycerol (DAG) after 15 s upon stimulation with ATP. The rank order of potency of a series of ATP and ADP analogues for stimulation of inositol trisphosphate (InsP3) formation was ATP greater than ATP gamma S greater than beta gamma-methylene-ATP greater than beta gamma-imido-ATP greater than ADP, while ADP beta S, AMP, adenosine and GTP were inactive, indicating the presence of P2y-purinergic receptors. ATP also stimulated a marked synthesis of prostaglandin E2 (PGE2). The rank order of potency of different ATP and ADP analogues was identical to that of InsP3 generation. Pre-treatment of the cells with pertussis toxin strongly attenuated ATP-induced formation of InsP3 and DAG. Short-term (10 min) pre-treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, produced a dose-dependent inhibition of the ATP-stimulated InsP3 generation. Furthermore, inhibition of protein kinase C by the potent inhibitor staurosporin, or downregulation of protein kinase C by longterm (24 h) incubation of the cells with TPA, resulted in an enhanced formation of InsP3 towards a stimulation with ATP.     

Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.     

Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition

RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5'-O-(thiotriphosphate) (ATPgammaS). hRAD51 fully saturates with ATP/ATPgammaS regardless of DNA cofactor (K(D) approximately 5 microm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPgammaS binding (K(app1) approximately 3 microm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K(app2) > or = 125 microm; >1 ADP/1 hRAD51). We could detect ADP --> ATP exchange in the high affinity ADP binding mode (K(app1)) but not the low affinity binding mode (K(app2)). At low ATP concentrations (<0.3 mm), ADP and ATPgammaS competitively inhibit the hRAD51 ATPase (K(m)((app)) > K(m)). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPgammaS that were 20-fold above the K(D). Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially ( approximately 10-fold) and ATP modestly ( approximately 3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.     

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry

A method using ion-pairing liquid chromatography-mass spectrometry (MS) was developed for analyzing adenosine 5(')-monophosphate (AMP), adenosine 5(')-diphosphate (ADP), and adenosine 5(')-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization-MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0.1 to 20 microM for AMP, 2 to 20 microM for ADP, and 2.5 to 20 microM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120 microg/ml cause a gradual increased in energy charge potential of the cells.