Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

ATP potentiates competitive inhibition of guanylyl cyclase A and B by the staurosporine analog, Gö6976: reciprocal regulation of ATP and GTP binding

Natriuretic peptides and ATP activate and Gö6976 inhibits guanylyl cyclase (GC)-A and GC-B. Here, the mechanism of inhibition was determined. Gö6976 progressively increased the Michaelis-Menten constant and decreased the Hill coefficient without reducing the maximal velocity of GC-A and GC-B. In the presence of 1 mm ATP, the K_i was 1 μm for both enzymes. Inhibition of GC-B was minimal in the absence of ATP, and 1 mm ATP increased the inhibition 4-fold. In a reciprocal manner, 10 μm Gö6976 increased the potency of ATP for GC-B 4-fold. In contrast to a recent study (Duda, T., Yadav, P., and Sharma, R. K. (2010) FEBS J. 277, 2550–2553), neither staurosporine nor Gö6976 activated GC-A or GC-B. This is the first study to show that Gö6976 reduces GTP binding and the first demonstration of a competitive inhibitor of a receptor guanylyl cyclase. We conclude that Gö6976 reduces GTP binding to the catalytic site of GC-A and GC-B and that ATP increases the magnitude of the inhibition.     

The gating of nucleotide-sensitive K+ channels in insulin-secreting cells can be modulated by changes in the ratio ATP4−/ADP3− and by nonhydrolyzable derivatives of both ATP and ADP

Summary The³¹P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K⁺ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K⁺ channels, even at ATP^4–/ADP^3– concentration ratios greater than 71. Interactions between ATP^4– and ADP^3– seem competitive. An increase in the concentration ratio ATP^4–/ADP^3– consistently evoked a decrease in the open-state probability of K⁺ channels; conversely a decrease in ATP^4–/ADP^3– increased the frequency of K⁺ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP^4– and ADP^3–, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP--S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K⁺ channels, in the presence of ATP. Similarly, ADP was able to activate K⁺ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and methylene ATP.     

Mechanism of inhibition of mitochondrial ATP synthase by 17β−Estradiol

17β-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling (“slipping”) of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and V_FCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and V_olig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the F_o component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F₁-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Generation and Characterization of ATP Analog-specific Protein Kinase Cδ

To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (K_m) and 150 times higher efficiency (k_cat/K_m) than wild type (WT) PKCδ toward N⁶-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N⁶-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N⁶-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N⁶-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates.     

Synthesis and reactivity of novel γ-phosphate modified ATP analogues

We hereby present a simple yet novel chemical synthesis of a family of γ-modified ATPs bearing functional groups on the γ-phosphate that are amenable to further derivatization by highly selective chemical manipulations (e.g., click chemistry, Staudinger ligations). A preliminary screen of these compounds as phosphate donors with a typical wild type protein kinase (cdk2) and one of its known substrates p27^kip1 is also presented.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Structure and Function of ε-Subunit of ATP Synthase in Higher Plant Chloroplast

Theε-subunit is the smallest subunit of chloroplast ATP synthase, and is known to inhibit ATPase activity in isolated CF1-ATPase. As a result ε is sometimes called an inhibitory subunit. In addition, and perhaps more importantly, theε-subunit is essential for the coupling of proton translocation to ATP synthesis (as proton gate). The relation between the structure and function ofε-subunit of ATP synthase in higher plant chloroplast has been studied by molecular biological methods such as site-directed mu-tagenesis and truncations for C- or N-terminus ofε-subunit. The results showed that: (1) Thr42 ofε-subunit is important for its structure and function; (2) compared with theε-subunit in E.. coli, theε-subunit of chloroplast ATP synthase is more sensitive to C- or N-terminus truncations.     

Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

Regulation of Cl− Secretion by Extracellular ATP in Cultured Mouse Endometrial Epithelium

The present study explored regulation of electrogenic ion transport across cultured mouse endometrial epithelium by extracellular ATP using the short-circuit current (I _SC ) and the patch-clamp techniques. The cultured endometrial monolayers responded to apical application of ATP with an increase in I _SC in a concentration-dependent manner (EC₅₀ at 3 μm). Replacement of Cl⁻ in the bathing solution or treatment of the cells with Cl⁻ channel blockers, DIDS and DPC, markedly reduced the I _SC , indicating that a substantial portion of the ATP-activated I _SC was Cl⁻-dependent. Amiloride at a concentration (10 μm) known to block Na⁺ channels was found to have no effect on the ATP-activated I _SC excluding the involvement of Na⁺ absorption. Adenosine was found to have little effect on the I _SC excluding the involvement of P₁ receptors. The effect of UTP, a potent P_2U receptor agonist on the I _SC was similar to that of ATP while potent P_2X agonist, α-β-Methylene adenosine 5′-triphosphate (α-β-M-ATP) and P_2Y agonist, 2-methylthio-adenosine triphosphate (2-M-ATP), were found to be ineffective. The effect of ATP on I _SC was mimicked by the Ca²⁺ ionophore, ionomycin, indicating a role of intracellular Ca²⁺ in mediating the ATP response. Confocal microscopic study also demonstrated a rise in intracellular Ca²⁺ upon stimulation by extracellular ATP. In voltage-clamped endometrial epithelial cells, ATP elicited a whole-cell Cl⁻ current which exhibited outward rectification and delayed activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The results of the present study demonstrate the presence of a regulatory mechanism involving extracellular ATP and P_2U purinoceptors for endometrial Cl⁻ secretion.     

Covalent modification of the non-catalytic sites of the H+-ATPase from chloroplasts with 2-azido-[α-32P]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H⁺-ATPase from chloroplasts, CF₀F₁, with 2-azido-[α-³²P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-³²P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-³²P]ATP was possible. CF₀F₁ with non-covalently bound 2-azido-[α-³²P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF₀F₁. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F₀-part and the F₁-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.     

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso-PC) on ATP and ρ-nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H+-ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso-PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso-PC was within 0-0.03%, and increased slightly when lyso-PC was higher than 0.03%. At the concentration of 0.03%, lyso-PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46μmol Pi·mg-1 protein·min-1 to 0.87 μmol Pi·mg-1 protein·min-1 while Km increased from 0.88 mmol/L to 1.15 mmol/L under lyso-PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0. Moreover, it was found lyso-PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso-PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso-PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C-terminal autoinhibitory domain in the plant plasma membrane H+-ATPase.     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.     

Cell damage caused by ATP depletion is reduced by magnesium and nickel in human fibroblasts--a non-specific calcium antagonism?

Calcium has been suggested to be the final common mediator of cell damage, but conflicting reports to prove this hypothesis have appeared. In order to elucidate the role of calcium in cell damage caused by ATP depletion, the effect of addition of calcium channel blockers (verapamil and nitrendipine) and non-specific antagonists (magnesium and nickel) was investigated in a model system of quiescent fibroblasts. ATP depletion was induced by metabolic inhibitors and the cell damage was assessed by the release of lactate dehydrogenase. Verapamil and nitrendipine did not protect the cells during ATP depletion, whereas a high concentration of Mg2+ (3-10 mmol/l) or a lower concentration of Ni2+ (0.5-1.0 mmol/l) reduced the cell damage considerably. An increased extracellular concentration of Ca2+ resulted in augmented cell damage. The effect of Mg2+ and Ni2+ was not due to an interference with the metabolic inhibitors or a reduction of the energy consumption. Both Ni2+ and Mg2+ were able to counteract the cell damage induced by entrance of Ca2+ after addition of the ionophore A23187. However, Mg2+ and Ni2+ were deleterious for the cells during ATP regeneration after an initial ATP decrease. These results indicate that a non-specific antagonism of Ca2+ may reduce cell damage, and, therefore, that Ca2+ may have an important role in cell damage, but also that a non-specific antagonism of Ca2+ during regeneration of ATP depleted cells is deleterious.     

Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase β-subunit gene family

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS–PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9 kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions.