Identification of peptides from the adenine binding domains of ATP and AMP in adenylate kinase: isolation of photoaffinity-labeled peptides by metal chelate chromatography.

Photoaffinity labeling with azidoadenine nucleotides was used to identify peptides from the ATP and AMP binding domains on chicken muscle adenylate kinase. Competition binding studies and enzyme assays showed that the 8-azido analogues of Ap4A and ATP modified only the MgATP2- site of adenylate kinase, whereas the 2-azido analogue of ADP modified the enzyme at both the ATP and AMP sites. The positions of the two nucleotide binding sites on the enzyme were deduced by isolating and sequencing the modified peptides. Photolabeled peptides were isolated by a new procedure that used metal chelate chromatography to affinity purify the photolabeled peptides prior to final purification by reverse-phase HPLC. The sequences of the peptides that were photolabeled with the 8-azido analogues corresponded to residues K28-L44, T153-K166, and T125-E135 of the chicken muscle enzyme. The residues that were present in both tryptic- and Staphylococcus aureus V-8 protease-generated versions of these peptides were assigned to the ATP binding domain on the basis of selective photoaffinity labeling with the 8-azidoadenine analogues. These peptides and an additional peptide corresponding to positions I110-K123 were photolabeled with 2-N3ADP. Since I110-K123 was photolabeled by 2-N3ADP but not by 8-N3Ap4A, it was assigned to the AMP binding domain.     

Analysis of adenine nucleotides at the picomole level with 32P-phosphoenolpyruvate and pyruvate kinase.

A new sensitive method for adenine nucleotide analysis is described. The key reaction is the phosphorylation of ADP by [³²P]PEP in a reaction catalyzed by pyruvate kinase, with the extent of transfer of ³²P to ADP being determined by adsorbing the nucleotides onto charcoal. The nonadenine nucleoside diphosphates which also react in the pyruvate kinase reaction are corrected for by determining the ³²P retained in the nucleotide fraction after a second incubation with hexokinase and excess glucose. ATP is determined as ADP, after it is quantitatively converted by hexokinase in the presence of excess glucose. Similarly, AMP is analyzed by its conversion to ADP in an incubation with excess ATP and adenylate kinase. The sensitivity of the method for ADP and ATP is 0.05–0.5 pmoles while for AMP it is 5 pmoles.     

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.     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Adenine nucleotide metabolism by chondrocytes in vitro: Role of ATP in chondrocyte maturation and matrix mineralization

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism. © 1995 Wiley-Liss Inc.     

Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents.

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.     

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

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.     

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.     

Human airway ecto-adenylate kinase. A mechanism to propagate ATP signaling on airway surfaces

Mechanically induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors. Ectoenzymes detected on these cells were recently shown to terminate ATP-mediated responses by sequential dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We now demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP + AMP 2ADP. This phosphotransferase exhibited a bilateral distribution on polarized primary cultures of human bronchial epithelial cells with a 4-fold higher activity on the mucosal surface. Ecto-AK presented an absolute requirement for magnesium and adenine-based nucleotides. UMP, GMP, and CMP could not substitute for AMP as gamma-phosphate acceptor, and UDP could not replace ADP. Apparent K(m) and V(max) values were 23 +/- 5 microM and 1.1 +/- 0.1 nmol x min(-1) x cm(-2) for ATP and 43 +/- 6 microM and 0.5 +/- 0.1 nmol x min(-1) x cm(-2) for ADP. Ecto-AK accounted for 20% of [gamma-(32)P]ATP dephosphorylation, and the impermeant AK inhibitor, diadenosine pentaphosphate, reduced ADPase activity by more than 70% on both epithelial surfaces. Time course experiments on ATP metabolism demonstrated that ecto-AK significantly prolongs effective ATP and ADP concentrations on airway epithelial surfaces for P2 receptor signaling and reduces by 6-fold adenosine production. Our data suggest a role for this nucleotide entrapment cycle in the propagation of purine-mediated mucociliary clearance on human airway epithelial surfaces.     

Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p

SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative splice sites. These enzymes are highly unique members of the protein kinase family. SRPKs contain a large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate specificity determinants. The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but binds ATP with a high K(m), suggesting that some of these distinctive structural features may be correlated with nucleotide binding [Aubol et al. (2002) Biochemistry 41, 10002-10009]. To address this issue, the nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy. The affinities of several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase, cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg(2+), using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP. This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.     

Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.     

A study of spatial and temporal variability in the adenine nucleotides of lake phytoplankton during a diel cycle (Lac Pavin — France)

A study of diel variations in the adenine nucleotide concentrations of natural phytoplanktonic populations led to the following conclusions:There are significant diel variations in cellular ATP concentrations. However, this temporal variability is not as great as the spatial variability between the various sampling depths, characterized by different phytoplankton populations. Within a globally stable pool of adenine nucleotides, diel variations inside the algal cell mainly take place at the expense of ATP and AMP, while ADP concentrations remain relatively stable.The statistical relationship between algal biomass obtained from cell counts and from adenine nucleotides also confirms maximum stability for ADP and (ATP + ADP + AMP). The diel variations in adenine nucleotide cell concentrations gradually become smaller with depth. This probably reflects the importance of light in causing such variability.     

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.     

Extracellular Interconversion of Nucleotides Reveals an Ecto-Adenylate Kinase Activity in the Rat Hippocampus

Here, the extracellular interconversion of nucleotides and nucleosides was investigated in rat hippocampal slices and synaptosomes by an HPLC-UV technique. Adenosine 5′-triphosphate (ATP) was converted to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), adenosine, inosine, and hypoxanthine in the slices, whereas ADP elicited parallel and concentration-dependent formation of ATP and AMP. The specific adenylate kinase inhibitor diadenosine pentaphosphate decreased the rate of decomposition of ADP and inhibited the formation of ATP. No substantial changes in the interconversion of ADP to ATP and AMP were found in the presence of dipyridamole, flufenamic acid, the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium (PPADS), and the alkaline phosphatase substrate para-nitrophenylphosphate. Negligible levels of nucleotides were generated when uridine 5′-diphosphate (UDP), AMP or adenosine were used as substrates. Ecto-adenylate kinase activity was also observed in purified synaptosomes. In summary, we demonstrate the presence of an ecto-adenylate kinase activity in the hippocampus, which is a previously unrecognized pathway that influences the availability of purines in the central nervous system.     

Antagonistic regulation of native Ca2+- and ATP-sensitive cation channels in brain capillaries by nucleotides and decavanadate

Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 microM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3'-5' cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels.     

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.     

Effects of Salinity, Diurnal Cycle and Age on Nucleotide Pools of Bean Leaves

In the present study, we investigated the relationship betweensalt stress and nucleotide levels in the shoot of Phaseolusvulgaris L. cv. Stringless Green Pod to determine if reducedgrowth was correlated with reduced nucleotide levels. Overallfresh weight of 25-d-old plants after having been on full salttreatment for 7 d was 33% lower compared with untreated plants.Shoot fresh weight decreased by 40% compared with 22% for theroots thus increasing the root to shoot ratio from 0·7to 0·9. We examined young and juvenile leaves as wellas mature leaves in order to compare growing tissue to fullyenlarged tissue. To ascertain whether the effects of salt stresson nucleotide pools were more severe during the day than atnight, we studied the combined effects of diurnal cycle andsalt stress on these nucleotide pools. Salt treatment selectivelyaffected certain nucleotide pools with the adenine nucleotides(AdN) being the most affected. We found large diurnal fluctuationsof AdN pools in all leaves. During the day, AMP and ADP increasedwhile ATP decreased. The sum, ATP + ADP, tended to remain constantand in mature leaves total AdN increased with AMP, an indicationof net synthesis. At night, ATP increased in all leaves. However,salt stress prevented this night-time increase in mature leaveswhile enhancing it in juvenile and young leaves. In the daytime,salt stress caused a nearly 2-fold increase in AMP of youngleaves and a large increase in the adenylate kinase mass actionratio (K). At night, the excess AMP disappeared with no changein total AdN. It is clear from these results that salt stressdid not reduce shoot growth by depleting ATP in growing leaves.It did, however, reduce the ATP level of mature leaves and perhapstheir ability to supply essential metabolites for growing regions. Key words: Phaseolus, nucleotides, salt stress, salinity, growth     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.