Multinuclear NMR study of the interaction of vanadate with mononucleotides, ADP, and ATP

The interaction of vanadate with 5'-mononucleotides, ADP, ATP, and various molecules containing some of their chemical moieties was studied in aqueous solution in the pH region of 5-9 using proton, 13C, 31P, and 51V nuclear magnetic resonance (NMR) spectroscopy. All the compounds studied formed noncyclic vanadate esters through interaction of monovanadate or divanadate with the hydroxyl groups of the ribose ring. Noncyclic anhydrides were also formed with the phosphate groups of ribose 5-phosphate, the mononucleotides, ADP, ATP, phosphate, pyrophosphate, and tripolyphosphate. In particular, ADP and ATP analogs resulted from AMP (AMPV and AMPV2) and from ADP (ADPV). Cyclic esters of trigonal bipyramidal geometry resulted from the interaction of vanadate with two ribose ring cis hydroxyl groups. AMP, CMP, and UMP formed two such complexes of 1:1 and 1:2 stoichiometries, similar to what has been observed for uridine and other nucleosides. However, 2'-deoxy-AMP does not yield this type of complexes. ADP and ATP also form similar cyclic ester complexes with vanadate, which does not chelate their pyrophosphate and tripolyphosphate moieties. Nevertheless, the separate pyrophosphate (PP) and tripolyphosphate (PPP) ligands form cyclic anhydrides of octahedral geometry with vanadate. However, their binding to vanadate is weaker than that of the ribose ring of nucleotides. Competition experiments between ethylene glycol and phosphate (P), pyrophosphate (PP), or tripolyphosphate (PPP) show that the relative strength of the interaction of these ligands with vanadate is PP greater than ethylene glycol greater than PPP greater than P.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Guanosine triphosphate (GTP): the major organic phosphate in the erythrocytes of the elasmobranch Mustelus canis (smooth dogfish).

1. Ion exchange column chromatography of red cell extracts from the smooth dogfish (Mustelus canis) reveals a phosphate pool which includes inorganic phosphate, AMP, ADP and ATP. 2. GTP and an iron complex of this nucleotide (Fe-GTP) account for approximately 40% of the principal organic phosphates. 3. ATP accounts for 30%, and AMP and ADP approximately 10% each. 4. 2,3-DPG is not detectable by either enzymatic or chromatographic analysis. 5. The use of a "purified" buffer system consisting of redistilled formic acid and de novo synthesized ammonium formate increases the ratio of GTP to Fe-GTP from approximately 1.4:1 to 10:1. This suggests that all of the iron nucleotide complexes (ATP and GTP) previously reported in red cells may not represent a normally occurring intraerythrocyte complex.     

Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds

The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP.     

Interaction centres of purine nucleotides: adenosine-5'-diphosphate and adenosine-5'-triphosphate in their reactions with tetramines and Cu(II) ions

The interactions between the nucleotides: adenosine-5'-diphosphate (ADP) and adenosine-5'-triphosphate (ATP) with spermine (Spm) and 1,11-diamine-4,8-diazaundecane (3,3,3-tet), as well as Cu(II) ions are studied. In the metal-free systems nucleotide-polyamine molecular complexes have been found to form, in which the interaction centres are the nitrogen atoms of the purine ring N(1) and N(7), oxygen atoms of the phosphate group of the nucleotide (for 3,3,3-tet) and protonated nitrogen atoms of the polyamine. Significant differences in the mode of metallation between the systems with Spm and 3,3,3-tet have been established. In the systems with Spm, the main products are protonated species with [N(7),O] chromophore and the nitrogen N(1) is involved in the intramolecular interaction additionally stabilising the complex. In the systems with 3,3,3-tet the formation of metal-ligand-ligand (MLL) species has been observed, in which the oxygen atoms from the phosphate group and the nitrogen atoms from the polyamine are involved in the metallation, while the N(1) and N(7) atoms from the purine ring of the nucleotide remain outside the inner coordination sphere of the copper ion. The main centre of metallation in the nucleotide, both with Spm and 3,3,3-tet, is the phosphate group of the nucleotide.     

How does the mitochondrial ADP/ATP carrier distinguish transportable ATP and ADP from untransportable AMP and GTP?Dynamic modeling of the recognition/translocation process in the major substrate binding region

To understand the transport mechanism of the bovine heart mitochondrial ADP/ATP carrier at the atomic level, we studied the four-dimensional features of the interaction of various purine nucleotides with the adenine nucleotide binding region (ABR) consisting of Arg(151)-Asp(167) in the second loop facing the matrix side. After three-dimensional modeling of ABR based on the experimental results, its structural changes on interaction with purine nucleotides were examined by molecular dynamics computation at 300 K. ATP/ADP were translocated to a considerable degree from the matrix side to the inner membrane region accompanied by significant backbone conformational changes, whereas neither appreciable translocation nor a significant conformational change was observed with the untransportable nucleotides AMP/GTP. The results suggested that binding of the terminal phosphate group and the adenine ring of ATP/ADP with Arg(151) and Lys(162), respectively, and subsequent interaction of a phosphate group(s) other than the terminal phosphate with Lys(162) triggered the expansion and subsequent contraction of the backbone conformation of ABR, leading to the translocation of ATP/ADP. Based on a simplified molecular dynamic simulation, we propose a dynamic model for the initial recognition process of ATP/ADP with the carrier.     

Heterotropic interactions of ligands with phosphorylase b.

1. The interaction of rabbit muscle glycogen phosphorylase b with pairs of ligands has been examined. 2. The electron spin resonance spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. 3. AMP competes with a series of nucleotides (ADP, ATP, CMP aand cytosine) but with adenosine a ternary enzyme - AMP - adenosine complex can be formed. 4. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. 5. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. 6. The effect of glucose b-phosphate on phosphorylase is rather complex as it 'formally competes' with both AMP and UDPG probably mediated by a conformational changes and not by 'direct' interactions with these two ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.     

Complexation and phase transfer of nucleotides by gramicidin S

Gramicidin S (GrS), an amphiphilic cyclosymmetric decapeptide produced by Bacillus brevis G-B and Nagano, binds nucleotides in water to yield a complex which partitions into organic solvents. The observed phase-transfer efficiencies at a given pH increase in the order AMP less than ADP less than ATP. The lipophilic complexes have well-defined stoichiometries, which were determined to be 1:1 for ADP-GrS at pH 7 and ATP-GrS at pH 3 and 1:2 for ATP-GrS at pH 7. The interaction is primarily ionic, involving coordination of the ornithine N delta H3+ groups of the peptide and the phosphoryl groups of the nucleotide, with little contribution from the nucleoside moiety. Exchange of organic and inorganic phosphates was also found to be mediated by GrS. The nucleotide complexes are sparingly soluble in water and self-associate extensively in CHCl3, most likely by cross-beta-aggregation, to yield large, ribbonlike aggregates which give rise to broad NMR resonances. Structures for the 1:1 and 1:2 complexes are proposed. In the latter, two GrS molecules envelop the nucleotide, orienting their apolar faces externally in opposite directions, while the lateral faces retain considerable polar character and direct aggregation in organic media. The 1:1 complex possesses a single apolar face and is less lipophilic. Binding constants were estimated by simulation of the extraction data. For the 1:1 complexes, K1:1 congruent to 4 X 10(4) M-1 for either ADP or ATP. Phase transfer of the ATP complex at pH 7 could be modeled either by stochastically independent binding to two noninteracting sites on the nucleotide with K1 approximately K2 approximately K1:1 or by a sequential process with K1 approximately K1:1 and K2/K1 less than 100. It is concluded that the apparent selectivity of GrS for ATP over ADP is a consequence of the greater lipophilicity and tendency to aggregate of the 1:2 complex, rather than an intrinsically higher binding affinity for triphosphates. GrS is, to our knowledge, the first peptide known to possess phase-transfer activity toward nucleotides; this is, in addition, the first molecular recognition process in which GrS is demonstrated to participate in vitro at physiologically active concentrations.     

On the metal-ion coordinating properties of the 5'-monophosphates of 1, N6-ethenoadenosine (epsilon-AMP), adenosine and uridine. Comparison of the macrochelate formation in the complexes of epsilon-AMP, AMP, ADP and ATP

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP2-) has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 2.5 +/- 0.3 M-1, is within experimental error identical to the value determined earlier for AMP2-,K = 2.1 +/- 0.4 M-1. The conditions for the potentiometric pH titrations, used to determine the acidity constants of H2(epsilon-AMP), H2(AMP), and H(UMP)- and the stability constants of the metal ion (M2+) complexes of the corresponding nucleoside 5'-monophosphates (NMP), were chosen so that the ligands were present in the monomeric form. The stabilities of Mg(epsilon-AMP) and Mg(AMP) are similar; however, the stabilities of the Mn2+, Cu2+ and Zn2+ complexes of epsilon-AMP2- are much larger (in the case of Cu2+ by a factor of 700) than those of AMP2-. This is due to the much larger metal ion affinity of the epsilon-adenosine moiety compared to that of the parent adenosine residue. As the uridine moiety does not participate in complex formation, the stability constants of M(UMP) have been used to evaluate the extent of macrochelation (i.e. the simultaneous coordination of M2+ to the base moiety and the phosphate group) in the epsilon-AMP and AMP complexes: the concentration of the macrochelated isomer is considerably larger for M(epsilon-AMP) than for M(AMP). A comparison with previous results for the complexes with ADP3- and ATP4- indicates the order, M(AMP)cl less than M(ADP)-cl greater than M(ATP)2-cl for the tendency to form macrochelates (cl). Due to the relatively high affinity of the epsilon-adenosine moiety towards Mn2+, Cu2+ and Zn2+, the phosphate-monoprotonated complexes M(H . epsilon-AMP)+ also become important; the corresponding complexes play only a minor role in the M2+/AMP systems. Intramolecular aromatic-ring stacking occurs in the ternary Cu(2,2'-bipyridyl)(NMP) complexes: about 80% of Cu(Bpy)(AMP) and Cu(Bpy)(epsilon-AMP) exist as the stacked isomer in aqueous solution; for the former system it has been shown in a previous X-ray study that the intramolecular ligand-ligand interaction occurs also in the solid state [Aoki, K. (1978) J. Am. Chem. Soc. 100, 7106]. Overall, the results emphasize that great care should be exercised in drawing conclusions based on studies of metal-ion-containing enzymic systems in which the natural adenine nucleotide cofactors have been replaced by the corresponding 1,N6-etheno derivatives.     

Stimulatory effect of phosphate, ATP and ADP on iron transfer from Fe(III)-bleomycin to apotransferrin

1. The rate of ferric ion transfer from Fe(III)-bleomycin to apotransferrin was increased in the presence of orthophosphate, ATP and ADP, while AMP was without effect. 2. Ortho phosphate activation probably involves formation of a Fe(III)-bleomycin-phosphate complex. The optical absorption of Fe(III)-bleomycin at 450 nm is enhanced in the presence of phosphate. 3. ATP and ADP remove the ferric ion from the iron-drug complex; thus making the ferric ion readily available for uptake by apotransferrin. 4. Low concentrations of ATP, ADP and AMP, also enhance the 450 nm absorption of the iron-drug complex. Higher ATP and ADP concentrations reduce both the 450 and 384 nm absorption of Fe(III)-bleomycin.     

Tightly bound nucleotides affect phosphate binding to mitochondrial F1-ATPase

The interaction of inorganic phosphate with native and nucleotide-depleted F1-ATPase was studied. F1-ATPase depleted of tightly bound nucleotides loses the ability to bind inorganic phosphate. The addition of ATP, ADP, GTP and GDP but not AMP, restores the phosphate binding. The nucleotides affecting the phosphate binding to F1-ATPase are located at the catalytic (exchangeable) site of the enzyme. The phosphate is thought to bind to the same catalytic site where the nucleotide is already bound. It is thought that ADP is the first substrate to bind to F1-ATPase in the ATP synthesis reaction.     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.     

Structural insight into AMPK regulation: ADP comes into play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.     

Interactions of 1,12-diamino-4,9-dioxadodecane (OSpm) and Cu(II) ions with pyrimidine and purine nucleotides: adenosine-5'-monophosphate (AMP) and cytidine-5'-monophosphate (CMP)

The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A potentiometric method was applied to determine the composition and stability constants of complexes formed, while the mode of interactions was analysed by spectral methods (ultraviolet and visible spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), (13)C NMR, (31)P NMR). In metal-free systems, molecular complexes nucleotide-polyamine (NMP)H(x)(OSpm) were formed. The endocyclic nitrogen atoms of the purine ring N(1), N(7), the nitrogen atom of the pyrimidine ring N(3), the oxygen atoms of the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine were the reaction centres. The mode of interaction of the metal ion with OSpm and the nucleotides (AMP or CMP) in the coordination compounds was established. In the system Cu(II)/OSpm the dinuclear complex Cu(2)(OSpm) forms, while in the ternary systems Cu(II)/nucleotide/OSpm the species type MH(x)LL' and MLL' appear. In the MH(x)LL' type species, the main centres of copper (II) ion binding in the nucleotide are the phosphate groups. The protonated amino groups of OSpm are involved in non-covalent interaction with the nitrogen atoms N(1), N(7) or N(3) of the purine or pyrimidine ring, whereas at higher pH, deprotonated nitrogen atoms of polyamine are engaged in metallation in MLL' species.     

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.     

Substrate specificity of T4 RNA-ligase. The role of phosphate nucleotide residues in the formation of a covalent AMP-RNA-ligase complex]

The inhibiting effect of adenosine, AMP, ADP, ATP, gamma-thio ATP (I), beta,gamma-imine ATP (II), beta,gamma-methylene ATP (III), P1,P3-di(adenosine-5') triphosphate (IV), P1,P4-di(adenosine-5') tetraphosphate (V) and adenosine 5'-tetraphosphate (VI) on the first step of the T4 RNA ligase reaction was studied. All the compounds tested, with the exception of adenosine, appeared to be competitive inhibitors of the first step of the enzymatic reaction. The inhibition constants (Ki) for the ATP analogs were determined. The data obtained suggest that the efficiency of inhibition depends on the number of phosphate groups and on the structure of ATP analogs. All the compounds under study (I-VI), except for AMP and ADP, form covalent AMP-RNA ligase complexes.     

Non-enzymatic ATP-mediated binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA

ATP mediates covalent binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA. This non-enzymatic reaction has been studied with 6-[14C]hydroxymethylbenzo[alpha]pyrene (]14C]BP-6-CH2OH) and 7-[14C]-hydroxymethylbenz[alpha]anthracene ([14C]BA-7-CH2OH) at 37 degrees C in Tris buffer (pH 7.0). While ADP mediates the reaction 25-50% as well as ATP, six other possible phosphate donors including AMP were inactive as cofactors. A complex response to ATP occurred in which low binding of BP-6-CH2OH or BA-7-CH2OH was observed at concentrations of ATP below 2.5 mM, but a greater than linear response to higher concentrations of ATP was observed until ATP was saturating. Binding of the substrates to RNA was much lower than to DNA. Fluorescence spectra of BP-6-CH2OH bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[alpha]pyrene bound to DNA and free 6-methylbenzo]alpha]pyrene, indicating that ATP-mediated binding of BP-6-CH2OH to DNA occurs at the 6-methyl group. The fate of ATP and ADP in the binding reaction of BP-6-CH2OH was examined by thin layer chromatography. Loss of one phosphate group occurs during the reaction. With ATP the rate of loss is about 100-fold greater than the rate of binding of BP-6-CH2OH to DNA. This implies that the binding reaction proceeds through formation of a presumed reactive and unstable phosphate ester intermediate which then inefficiently binds to DNA.     

AMP deamination delays muscle acidification during heavy exercise and hypoxia

In silico studies carried out by using a computer model of oxidative phosphorylation and anaerobic glycolysis in skeletal muscle demonstrated that deamination of AMP to IMP during heavy short term exercise and/or hypoxia lessens the acidification of myocytes. The concerted action of adenylate kinase and AMP deaminase, leading to a decrease in the total adenine nucleotide pool, constitutes an additional process consuming ADP and producing ATP. It diminishes the amount of ADP that must be converted to ATP by other processes in order to meet the rate of ADP production by ATPases (because the adenylate kinase + AMP deaminase system produces only 1 ATP per 2 ADPs used, ATP consumption is not matched by ATP production, and the reduction of the total adenine nucleotide pool occurs mostly at the cost of [ATP]). As a result, the rate of ADP consumption by other processes may be lowered. This effect concerns mostly ADP consumption by anaerobic glycolysis that is inhibited by AMP deamination-induced decrease in [ADP] and [AMP], and not oxidative phosphorylation, because during heavy exercise and/or hypoxia [ADP] is significantly greater than the Km value of this process for ADP. The resultant reduction of proton production by anaerobic glycolysis enables us to delay the termination of exercise because of fatigue and/or to diminish cell damage.     

P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.