Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Study of carnosine complexes with nucleotides by the method of high resolution nuclear magnetic resonance]

It is shown that the formation of a carnosine--nucleotide complex (ATP, ADP, AMP) takes place. The stability of the complex mainly depends on: 1) the staking interaction between the heterocyclic rings of carnosine and nucleotides; 2) the electrostatic interaction between the phosphate groups of nucleotide and the positive charged amino group NH3+ of the beta-alanine part of carnosine. The formation of the hydrogen bond between dipeptide COO- group and N1 or N7 of nucleotide is also possible. The complex stability strongly depends on the charge-state of the components and little on the number of the phosphate groups of nucleotide (ATP greater than or equal to ADP greater than AMP).     

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.     

Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

Effect of adenine nucleotides and pyrophosphate on the exchange of transferrin-bound carbonate.

1. ATP, ADP and pyrophosphate accelerate the exchange of carbonate of the transferrin-iron-carbonate ternary complex, while AMP, cyclic AMP and phosphate have no effect. 2. ATP promotes carbonate exchange without removing iron from transferrin, whereas pyrophosphate effectiely attacks both the anion and iron components of the ternary complex. 3. Transferrin readily takes over iron from its ATP or pyrophosphate complex. 4. Neither ATP nor pyrophosphate can substitute for carbonate of the ternary complex. These results fit in well with the concept that ATP may play a direct role in the iron uptake by reticulocytes.     

Post-ischaemic synchronous purine nucleotide oscillations in perfused rat heart

Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.     

Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F(1)F(0) ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F(1)F(0) ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F(1)F(0) ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.     

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.     

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.     

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate)

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.     

Role of ADP in the control of actomyosin superprecipitation and ATPase activity.

ATP, in the presence of 0.05–0.15 m KCl and greater than 50 μm Mg²⁺, induces dissociation (clearing) followed by superprecipitation of skeletal muscle actomyosin. Superprecipitation has been studied as a model of muscle contraction, and ATP depletion has been associated with the onset of superprecipitation. Recent studies [Puszkin and Rubin (1975) Science188, 1319–1320] indicate that ADP stimulates superprecipitation without increasing the rate of ATP hydrolysis. We confirm that ADP stimulates superprecipitation; however, contrary to the experience of these investigators, ADP does stimulate ATP hydrolysis in the system studied here. We present evidence that superprecipitation is associated with generation of a critical ADP:ATP ratio but it appears that this ratio is an indirect measure of an associated but uncharacterized phenomenon which signals the onset of superprecipitation. Added ADP decreased the extent and duration of clearing, increased the rate of ATP hydrolysis, and increased the extent of superprecipitation of rat skeletal muscle actomyosin in the presence of excess Mg²⁺. The ADP effect was not mimicked by EDTA or AMP. The duration of clearing was related not to the time required to attain a specific level of any nucleotide phosphate, but to the time required to generate an ADP:ATP ratio of approximately 3.6. Apparently only that ADP generated in the system by ATP hydrolysis was involved in the critical ADP:ATP ratio. Added ADP stimulated myosin ATPase activity in 1.6 or 3.2 mm Mg²⁺. This effect was not mimicked by EDTA or AMP. The results are used to relate studies by others of myosin sulfhydryl modification to a recent model [Burke et al. (1973) Proc. Nat. Acad. Sci. USA70, 3793–3796] in which myosin MgATPase activity is inhibited by formation of a stable cyclic complex of MgATP and the S₁ and S₂ sites of heavy meromyosin.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

Coexistence of two ATP sites on the ouabain-complexed (Na+ + K+)-ATPase

When the effects of varying concentrations of ATP on the dissociation rate of the ouabain-enzyme complex were studied, the dissociation rate constant increased with increasing ATP concentrations up to 1 mM, and then decreased with further rise in ATP; indicating that ATP binds to two distinct sites on the complex. ADP and AMP-PNP had similar biphasic effects. GTP, CTP, UTP, and AMP-PCP reduced the dissociation rate. AMP and Pi had no effects. Increase in dissociation rate caused by 0.5 mM ATP was not abolished by saturating CTP, indicating the binding of CTP to only one of the two ATP sites. The data suggest the existence of separate catalytic and regulatory sites, with different affinities and nucleotide specificities.     

Effect of adenine nucleotides on the activator function of streptokinase

The addition of ATP or 3,5-AMP (but not UTP, GTP, CTP, AMP, 2,3-AMP, ADP, inorganic pyrophosphate) at a final concentration of 10(-1) M into streptokinase solution, pH 7.0 or 9.5, causes a dramatic inhibition of streptokinase-induced fibrinolysis. The specificity of ATP effect is fully lost at pH 3.0, when all nucleotides completely inhibit the activating function of streptokinase. Ribose-5-phosphate causes a similar effect at pH 3.0. The character of nucleotide action on the activating function of streptokinase considerably differs from their influence on proteolytic reactions.     

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.     

The activity of phosphate-dependent glutaminase from the rat small intestine is modulated by ADP and is dependent on integrity of mitochondria

The effect of adenine nucleotides and phosphate on rat small intestine phosphate-dependent glutaminase (PDG) activity was investigated in intact mitochondria. Disruption of the integrity of mitochondria by sonication or freeze-thawing resulted in loss of enzyme activity. ADP was the strongest adenine nucleotide activator of the enzyme giving a V_max that was over 5-fold of that for AMP or ATP. The sigmoid activation curve of PDG by ADP became hyperbolic in presence ATP. ADP also lowered the K_m for glutamine and increased V_max and these effects were further enhanced by the presence of ATP. Activation of PDG by phosphate and ADP was not completely additive suggesting some antagonism between the activators. There was no clear relationship between changing ATP/ADP ratios and PDG activity in presence of a constant concentration of phosphate. However, ratios of approximately 1:4 and 4:1 gave the highest and lowest activities, respectively. The pH dependence of PDG activity was affected by phosphate concentration and results suggest that the divalent ion is the activating species.     

Interaction of fluorescent 3'-[1,5-(dimethylamino)naphthoyl]adenine nucleotides with the solubilized ADP/ATP carrier

The binding of the 3'-[1,5-(dimethylamino)naphthoyl] (DAN) derivatives of AMP, ADP, and ATP to the solubilized ADP/ATP carrier is studied, evaluating primarily the fluorescence enhancement and 3H-labeled compound binding. DAN nucleotides also fluoresce when adsorbed to Triton X-100 micelles that are used for solubilization of the carrier. The partition of DAN-AMP between water and Triton X-100 micelles is measured, and it is shown to be shifted toward a higher content in Triton micelles with increasing salt concentration. In order to maintain a low level of fluorescence, the Triton content is decreased. The fraction of DAN nucleotide fluorescence due to carrier binding is determined by the suppression with bongkrekate (BKA). In contrast to the membrane-bound carrier, the solubilized preparation shows an increase of total BKA-sensitive fluorescence by 30-60% upon addition of ATP or ADP. In the solubilized atractylate-protein complex, the ADP-stimulated fluorescence amounts even to 80%. The suppression of fluorescence by BKA is independent of the presence of ADP or ATP, while that by carboxyatractylate (CAT) depends on ADP or ATP. The quantitation with [3H]BKA and [3H]CAT of these ligand interactions with DAN-AMP fluorescence shows that DAN-AMP fluorescence reflects the "m"-state carrier population and its redistribution under the influence of ADP or ATP. Thus, besides the "c"/"m" distribution, the kinetics of the c to m transition in the solubilized carrier also can be determined. The m share is increased to 80% when SO4, Pi, or pyrophosphate is present during solubilization. The rate of the ADP- or ATP-stimulated transition to the m state is markedly dependent on pH and on the presence of various anions, whereas the extent is little varied. The affinity decreases 4-fold going from DAN-AMP to DAN-ADP and to DAN-ATP (KD = 0.9, 1.6, and 3.2 microM). Comparison with physical binding of [3H]DAN nucleotides shows that the fluorescence yield of bound DAN-AMP is about 1.4 times higher than that of bound DAN-ATP. DAN substitution causes more than a 100-fold affinity increase for AMP and a 50-fold increase for ADP or ATP, probably because of interaction of the DAN group with a hydrophobic niche. A less specific, low-affinity displacement of DAN nucleotides by GDP, ADP, GTP and ATP (Ki = 1-2 mM) probably reflects primarily the ionic interactions at the binding center.