Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and reaction studies with adenine nucleotides on purified coupling factor fromRhodospirillum rubrum

Equilibrium binding studies with ATPase isolated fromRhodospirillum rubum chromotophores have been carried out using gel filtration.Binding experiments with variable concentrations of [¹⁴C]ADP show a biphasic saturation curve. With a parameter fitting computer program the dissociation constants for two distinct binding sites are determined as 7×10^–6 and 9×10^–5 M, respectively. The enzymebound radioactivity is recovered as ADP (80–90%), and the rest is converted to AMP and ATP. In the free nucleotides a large amount of AMP (about 70%) is found in addition to ADP.Analogous binding experiments with [¹⁴C]ATP are monophasic. Most of the bound radioactivity can be identified as ADP showing a dissociation constant corresponding to the high affinity site. The pattern of the free nucleotides is the same as in the experiments with ADP.These results indicate three separate binding sites on the enzyme: a low and a high affinity site for ADP, and a site at which ATP hydrolysis takes place. The analysis of the nucleotides suggests for the ADP sites a phosphoryl group transfer to produce ATP and AMP. Various experiments exclude the contamination of the enzyme preparation with adenylate kinase.Part of this work has been published in theBook of Abstracts, 4th International Congress on Photosynthesis, Reading, U.K., 1977.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Thymidine phosphorylation in wheat: analysis of phosphate transfer from ATP to thymidine

Extracts of wheat (Triticum vulgare Vill. [Triticum aestivum L.] var. Lemhi) seedlings contain thymidine-phosphorylating activity with ATP, ADP, or AMP and nucleotide hydrolase activity (ATP → → AMP). The synthesis of [³²P]dTMP exclusively from [α-³²P]ATP with none detectable from [γ-³²P]ATP demonstrates the absence of thymidine kinase and the presence of nucleoside phosphotransferase as the only observable thymidine-phosphorylating enzyme.     

Temperature acclimation in brook trout muscle: Adenine nucleotide concentrations, phosphorylation state and adenylate energy charge

Summary Cold acclimation in fish is associated with an elevation in metabolic rate. The present study investigates the role of adenine nucleotides and related compounds in metabolic regulation following temperature acclimation. Brook trout (Salvelinus fontinalis) were acclimated for 10 weeks to either +4°C or +24°C. Both groups of fish were exercised at 2.5 body lengths s^–1 for 2 weeks prior to sacrifice in order to control for differences in spontaneous activity.Concentrations of ATP, ADP, AMP, P _i and PC were approximately 2-fold higher in white than red muscles. Temperature acclimation had little effect on total adenine nucleotide concentration in either muscle type. In white fibres acclimation to 4°C results in a 39% increase in [ADP] and [AMP], a 35% decrease in [PC] (phosphorylcreatine), and no significant change in [P _i ]. In contrast temperature has little effect on concentrations of these compounds in red muscle.Parameters of metabolic control — adenylate energy charge ([ATP]+0.5 [ADP]/[ATP]+[ADP]+[AMP]), phosphorylation state ([ATP]/[ADP]·[P _i ]), and the ratios [ATP][ADP] and [ATP][AMP] — were significantly lower in cold- than warm-acclimated white muscle. The observed changes in phosphorylation state and [ATP][AMP] are consistent with an increase in mitochondrial respiration and glycolysis, respectively.In conclusion, changes in metabolites may be an important factor in producing an enhanced metabolic rate in cold-acclimated fish.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

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.     

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline

1. Platelets containing adenine nucleotides labelled with ³H and ¹⁴C in vitro were aggregated biphasically with ADP and adrenaline. Amounts of ATP and ADP as well as the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine and adenine were determined in platelets and plasma at different stages of aggregation. 2. ATP and ADP were released during the second aggregation phase and had a low specific radioactivity compared with the ATP and ADP retained by the cells. The specific radioactivity of intracellular nucleotides increased during release. The parameters observed with ADP and adrenaline as release inducers were the same as for collagen and thrombin. 3. Release induced by all four inducers was accompanied by conversion of cellular [³H]ATP into extracellular [³H]-hypoxanthine. By variation of temperature, inducer concentration, time after blood withdrawal and use of acetylsalicylic acid, the aggregation pattern caused by adrenaline and ADP could be made mono- or bi-phasic. Release or second-phase aggregation was intimately connected with the ATP–hypoxanthine conversion, whereas first phase aggregation was not. 4. The [³H]ATP–hypoxanthine conversion started immediately after ADP addition. With adrenaline it usually started with the appearance of the second aggregation phase. The conversion was present during first phase of ADP-induced aggregation only if a second phase were to follow. 5. When secondary aggregation took place while radioactive adenine was being taken up by the platelets, increased formation of labelled hypoxanthine still occurred, but there was either no change or an increase in the concentration of labelled ATP. 6. Biphasically aggregated platelets converted [³H]adenine more rapidly into [³H]-ATP and -hypoxanthine than non-aggregated platelets. Addition of [³H]adenine at different stages of biphasic aggregation showed that more [³H]hypoxanthine was formed during than after the release step. 7. We conclude that ADP and adrenaline, like thrombin and collagen, cause extrusion of non-metabolic granula-located platelet adenine nucleotides. During release metabolic ATP breaks down to hypoxanthine, and this process might reflect an ATP-requiring part of the release reaction.     

Modulation of the chloroplast ATPase by tight binding of nucleotides

Inactivation of the chloroplast ATPase upon tight nucleotide binding was studied with several adenine nucleotide analogs. Compared with ADP, the other nucleoside diphosphates were less effective in the follwing order: IDP >?-ADP > 1-oxido-ADP > GDP. The nucleotide analogs compete with ADP for binding to the tight nucleotide-binding site(s) on the ATPase and also prevent further inactivation by ADP. AdoPP[NH]P also causes inactivation but has a lower affinity than ADP. [³H]GDP binds tightly to the ATPase, but the resulting enzyme-GDP complex is more readily dissociable than the enzyme-ADP complex. Although both nucleotides appear to bind to the same site, the catalytic and binding properties of the coresponding nucletide-enzyme complexes differ. Binding of GDP also decreases the rate and extent of the sontaneous decay of the activated enzyme. PP_i decreases the rate of inacivation caused by ADP and also the level of tigthly buond ADP. Based on these results, we suggest that two different confomations of the ATPase exist which contain tigthly bound ADP. The active conformation is conveted to the inactive conformation in the absence of a continued supply of energy by illumination or ATP hydrolysis.     

A simple, rapid preparation ofα-[32P]- labelled adenosine diphosphate

Hexokinase (EC 2.7.1.1) will convert commercially available α-[³²P]-labelled ATP into α-[³²P]-labelled ADP. A simple, rapid isolation procedure for the α-[³²P]-labelled ADP is described and this synthetic method can be used for the preparation of other α-[³²P]-labelled nucleoside diphosphates.     

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.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes Effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP

The effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP on isotope incorporation into phospholipids and the release of β-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3′ : 5′-monophosphate (0.1–1.0 mM) reduced β-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited ³²P_i incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-³H]inositol into the phosphoinositides. Added ATP (0.1–1.0 mM), but not other nucleotides, was found to potentiate β-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-³H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3′ : 5′-monophosphate at 2–4 mM concentration was not found to appreciably alter the incorporation of [γ-³²P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.     

Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit

The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.     

The substrate specificity of the human ADP/ATP carrier AAC1

Abstract

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [³²P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [¹⁴C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.     

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([γ-³²P]ATP and [5′,8-³H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.