ADP-ATP Exchange Research Articles

Chymotryptic cleavage of α-subunit in E 1-forms of renal (Na + + K +)-ATPase: effects on enzymatic properties, ligand binding and cation exchange

Chymotrypsin in NaCl medium at low ionic strength rapidly cleaves a bond in the N-terminal half of the α-subunit of pure membrane-bound (Na + + K +)-ATPase from outer renal medulla. Secondary cleavage is very slow and the α-subunit can be converted almost quantitatively to a 78 kDa fragment. The sensitive bond is exposed to cleavage when the protein is stabilized in the E 1 form by binding of Na + or nucleotides. The bond is protected in medium containing KCl (E 2 K form), but it is exposed when ADP or ATP are added (E 1KATP form). Fluorescence analysis and examination of ligand binding and enzymatic properties of the cleaved protein demonstrate that cleavage of the bond stabilizes the protein in the E 1 form with sites for tight binding of nucleotides and cations exposed to the medium. About two 86Rb ions are bound per cleaved α-subunit with normal affinity ( K d = 9 μM ). The bound Rb + is not displaced by ATP or ADP. The nucleotide-potassium antagonism is abolished and ATP is bound with high affinity both in NaCl and in KCl media. Na +-dependent phosphorylation is quantitatively recovered in the 78 kDa fragment, but the affinity for binding of [ 48V]vanadate is very low after cleavage. ADP-ATP exchange is stimulated 4–5-fold by cleavage; while nucleotide dependent Na +-Na +, K +-K +, or Na +-K + exchange are abolished. Cleavage with chymotrypsin in NaCl at the N-terminal side of the phosphorylated residue thus stabilizes the E 1 form of the protein and abolishes cation exchange and conformational transitions in the protein although binding of cations, nucleotides and phosphate is preserved. In contrast, cleavage with trypsin in KCl at the C-terminal side of the phosphorylated residue does not interfere with E 1-E 2 transitions and Na +-Na + or K +-K + exchange. This data support the notion that cation exchange and E 1–E 2 transitions are thightly coupled.

The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from ϱ + and ϱ o strains of saccharomyces cerevisiae

The energization of ϱ + and ϱ o isolated mitochondria was measured using a tetraphenylphosphonium selective electrode. In both strains, translocase mediated ATP ADP exchange energization was observed. This energization was more sensitive to uncoupler than that induced by respiration in ϱ + mitochondria. This observation is in accordance with the hypersensitivity of ϱ − cell growth to uncoupler.

Measurements of exchange in the reaction catalysed by creatine kinase using 14C and 15N isotope labels and the NMR technique of saturation transfer

31P-NMR measurements of saturation transfer have been used to measure exchange between the γ-phosphate of ATP and phosphocreatine and between the β-phosphate of ATP and the β-phosphate of ADP in the reaction catalysed by creatine kinase in vitro. The calculated exchange fluxes have been compared with measurements of 15N label exchange between creatine and phosphocreatine and 14C label exchange between ATP and ADP. At pH 8.0 the fluxes between phosphocreatine and the γ-phosphate of ATP and between the β-phosphates of ATP and ADP, measured by saturation transfer, were the same and equal, within experimental error, to the fluxes between creatine and phosphocreatine, measured by 15N label exchange, and between ADP and ATP, measured by 14C label exchange. At pH 7.0 the flux between phosphocreatine and the γ-phosphate of ATP, measured by saturation transfer, was equal, within experimental error, to the flux between creatine and phosphocreatine, measured by 15N label exchange. However, at low ADP concentrations (less than 0.2 mM), the flux between ATP and ADP measured by saturation transfer was significantly less than that between phosphocreatine and ATP and, more importantly, less than the ADP-ATP exchange flux measured by 14C label exchange. The saturation transfer and isotope exchange measurements at pH 7.0 have shown that it is valid to equate saturation transfer measurements of exchange between phosphocreatine and ATP in vivo with the potential for net chemical flux through the reaction. The observed discrepancy at pH 7.0 between the 14C and saturation transfer measurements of ATP ↔ ADP exchange can be explained if there is significant loss of saturation in an intermediate in the exchange reaction. Under these conditions analysis of the exchange according to two-site exchange model is invalid. In magnetisation transfer measurements of exchange in other enzyme catalysed reactions, the possible presence of a kinetically significant intermediate and therefore the validity of data analysis using a two-site exchange model should be considered.

Occlusion of Na + by the Na, K-ATPase in the presence of oligomycin

Oligomycin occludes Na+ in an E1-form of the Na,K-ATPase. The rate constants for the release of Na+ from the E1-form and for the transition to the E2-form are about 0.5 s-1. The effect of oligomycin is not seen using other cations which also have a Na+-like effect on the enzyme conformation. The inhibitory effect of oligomycin on the ADP-ATP dependent Na:Na exchange but not on the accompanying ADP-ATP exchange can be explained from a decrease in the rate of release of Na+ from an E1 approximately phosphoform with Na+ occluded, E'1 approximately P (Na3), i.e. with Na+ in the membrane phase, to an E"1 approximately PNa3 form with Na+ not occluded. E"1 approximately PNa3 is at a step before formation of E2-P, and disappears at a high rate when ADP reacts with E"1 approximately P (Na3).

The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. An analysis of ADP-induced oxygen jumps by linear nonequilibrium thermodynamics.

Uncertainty exists as to the proton stoichiometries of mitochondrial oxidative phosphorylation and consequently as to the ATP stoichiometries. In rat liver mitochondria, ADP/O ratios were determined from the total and extra oxygen consumed during ADP-stimulated respiration under conditions of quantitative conversion of ADP to ATP. For succinate, glutamate plus malate, 3-hydroxybutyrate, and 2-oxoglutarate, respectively, ADP/total O was 1.71, 2.71, 2.61, and 3.45. ADP/extra O was 2.03, 3.04, 3.23, and 4.15. The results were interpreted in terms of linear nonequilibrium thermodynamics. It was shown that ADP/extra O = Z/q where Z is the phenomenological stoichiometry and q is the degree of coupling. q was determined from the dependence of respiratory rate on delta Gp, the phosphorylation potential, and was about 0.98 for all substrates. The results were consistent with ideal ATP/O stoichiometries of 2 for succinate, 3 for glutamate plus malate, 3 or 3 1/4 for 3-hydroxybutyrate, and 4 for 2-oxoglutarate. Taking into account the oxidation-reduction free-energy changes measured across Sites 1 + 2 at static head (J.J. Lemasters, R. Grunwald, and R.K. Emaus J. Biol. Chem. 259, 3058-3063), an ideal ATP/O stoichiometry of 3 1/4 for 3-hydroxybutyrate is proposed. The lower ATP/O for glutamate plus malate is then accounted for by proton translocation linked to glutamate/aspartate exchange. The data suggest a new 13-proton scheme of chemiosmotic coupling in which proton stoichiometries are 3 for the F1Fo-ATPase, 1 for the exchange of ATP for ADP and Pi, and 5, 4, and 4 for Sites 1, 2, and 3.

Characterization of the exchange reactions of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase

The Fru-6-P/Fru-2,6-P 2 exchange reaction of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was almost entirely dependent on the presence of P i. This exchange was not due to a reversal of the bisphosphatase nor to trace amounts of adenine nucleotide in the enzyme. Exchange activity was maximal at pH 7, activated by ADP, and equal to 10–15 percent of the kinase V max. The ADP ATP exchange reaction was more resistant to various protein modifying agents than the kinase. These studies confirm the existence of both exchange reactions but do not prove they are related to the kinase reaction.

Limited proteolysis and photoaffinity labeling with 8-azido-ATP of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase.

Limited proteolysis and photoaffinity labeling of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase were studied. Proteolysis by trypsin proceeds in two stages in which the first cleavage yields a product, Mr about 53,000, which has lost 90% of fructose-6-P,2-kinase, but retains nearly 80% of fructose-2,6-bisphosphatase. Further digestion of this product yields a second cleavage product, Mr about 50,000, which is completely devoid of the kinase and most of the phosphatase activities. These results indicate that fructose-6-P,2-kinase resides only in the original ("native") enzyme (Mr = 55,000), but fructose-2,6-bisphosphatase activity is present in both the native enzyme and the cleavage product(s). All three activities of fructose-6-P,2-kinase including the forward, the reverse, and ATP-ADP exchange activities are lost to the same degree by the mild proteolysis. Ki of fructose-6-P for fructose-2,6-bisphosphatase is not altered by the proteolysis. Partial protection against the proteolysis is provided by ATP, fructose-6-P, and fructose-2,6-P2. When the tryptic digestion of fructose-6-P,2-kinase:fructose-2,6-bisphosphatase was performed before and after phosphorylation of the enzyme by cAMP-dependent protein kinase, both the first and the second cleavage products contained the phosphorylation site. 8-Azido-ATP serves as a substrate for fructose-6-P,2-kinase with a Km of about 1 mM. Exposure of the enzyme-8-azido-ATP complex results in covalent incorporation (0.7 mol/mol of subunit) and 90% inactivation of fructose-6-P,2-kinase without loss of fructose 2,6-bisphosphatase. When the native and the first cleavage product of tryptic digestion were photoaffinity labeled with [alpha-32P]8-azido-ATP, the radiolabel occurred only in the native enzyme. These results provide evidence in support of, although not conclusive, the idea that the active sites of this bifunctional enzyme are different and located in two distinct sites.

Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase.

The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6- bisophosphatase , were studied. Fructose-6-P,2-kinase catalyzes the isotope exchange of ADP with ATP in the absence of fructose-6-P at about 3% of the rate of the overall reaction. The Km values for ATP and ADP for the reaction are 120 and 66 microM, respectively. No isotope exchange between fructose-6-P with fructose-2,6-P2 in the absence of adenine nucleotide was observed. The ADP-ATP exchange is not inhibited by the presence of 25 microM (1.6 X Km) fructose-6-P and less than 20% inhibition was observed at higher concentrations. Fructose-6-P,2-kinase catalyzes the reversal of the reaction at a rate approximately one-half that of the forward direction. The fructose 2,6-bisphosphatase reaction is not inhibited by 1 to 20 mM ADP. Plots of the reciprocal of the concentration of either substrate versus the reciprocal of the concentration of the other substrate yield intersecting lines for both forward and reverse reactions. The Michaelis constants for ATP and fructose-6-P are 150 and 16 microM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 microM, respectively. ADP is a competitive inhibitor with respect to ATP with an inhibition constant of 0.6 mM. All other product inhibition patterns including ADP versus fructose-6-P and fructose-2,6-P2 versus ATP and fructose-6-P are noncompetitive. Fructose-6-P,2-kinase also catalyzes the hydrolysis of ATP at about 10% of the rate of the overall reaction. There is no detectable burst of ADP formation upon incubating ATP with a stoichiometric amount of the enzyme. Similarly no burst of fructose-6-P formation is observed by reacting fructose-2,6-P2 with fructose 2,6-bisphosphatase. These results demonstrate that (a) the fructose-6-P,2-kinase reaction is consistent with a sequential mode of substrate addition rather than a "ping-pong" mechanism, (b) phosphorylenzyme , if formed, is not likely a kinetically important intermediate, and (c) no common phosphorylenzyme intermedite between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.

Modification of the conformational equilibria in the sodium and potassium dependent adenosinetriphosphatase with glutaraldehyde.

Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.

Evaluation of the relationship between the intra- and extramitochondrial [ATP]/[ADP] ratios using phosphoenolpyruvate carboxykinase.

The ratio of free ATP to free ADP in the mitochondrial matrix [( ATPf]/[ADPf]) has been measured in suspensions of isolated mitochondria under conditions of active phosphorylation of extramitochondrial ADP. These measurements utilized phosphoenolpyruvate carboxykinase which is present in the matrix of mitochondria from the livers of guinea pigs, chickens, and pigeons. Mitochondria isolated from these sources also contain nucleoside diphosphate kinase, malate dehydrogenase, and glutamate dehydrogenase or 3-OH-butyrate dehydrogenase. Together these enzymes catalyze the synthesis of phosphoenolpyruvate and CO2 from oxaloacetate with oxidative phosphorylation as an energy source. These reactions have been shown to be fully reversible in suspensions of mitochondria isolated from the above sources. With oxidative phosphorylation as the source of ATP, phosphoenolpyruvate was synthesized from malate and conversely addition of phosphoenolpyruvate, ADP, and CO2 led to synthesis of malate and ATP. The forward and reverse reactions were allowed to continue until the rate of change of metabolite concentrations was minimal and then the latter were measured. The intramitochondrial [Mg-ATPf]/[MgADPf] was calculated from the equilibrium constants for the reactions and the measured steady state concentrations of the metabolites in both the intra- and extramitochondrial spaces. The value of the intramitochondrial [MgATPf]/[MgADPf] was found to exceed the extramitochondrial value (adjusted to the same free Mg2+ concentration) by a factor (+/- S.E.) of 0.83 +/- 0.22 (n = 17) for the forward reaction and 1.81 +/- 0.54 (n = 11) for the reverse reaction. It is concluded that the adenine nucleotide translocase catalyzes electroneutral exchange of ATP for ADP and that this reaction does not contribute significantly to the energetics of mitochondrial oxidative phosphorylation.

Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel

A new method for determination of 32 P i-ATP and [ 14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and P i by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and P i in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50–100 μl. Aliquots (2–6 μl) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and P i were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H +-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.

Reconstruction of steady state in cell-free systems: Interactions between glycolysis and mitochondrial metabolism: Regulation of the redox and phosphorylation states

A reconstituted “open” system comprising respiring mitochondria and actively glycolyzing muscle extract was devised for studies of vectorially mediated interactions. Glycogen particles were the substrate for the glycolyzing enzymes. Purified soluble (F 1) ATPase was added in varying quantities to establish a range of energetic steady states. The data generally confirm our recent conclusions (Wu and Davis, (1981) Arch. Biochem., Biophys. 208, 85–89) on the relative efficacy of the adenine nucleotides and their ratios, and of inorganic phosphate on flux through rate-controlling steps of glycolysis. When mitochondrial ATP synthesis was blocked, glycolytic flux was relatively rapid, and the lactate/pyruvate ratio increased with time to values up to greater than 300. If functional mitochondria were present, glycolytic flux was very strongly suppressed, provided the energy state ( ATP ADP ) was high, and the phosphate concentration[ Pi] was low. Adenine nucleotide control of glycolysis was to a large extent lost when the steady-state ATP ADP was below about 10, or if [ Pi] was elevated. In the two-phase system containing respiring mitochondria and components of the malate-aspartate shuttle, the ATP ADP and both extra- and intramitochondrial NAD +/NADH ratios were maintained constant, and to various perturbable levels with varying energy load (ATPase). The gradient in reduction potentials attained values (Δ G rmredox ) of up to about 2.5 kcal. The extramitochondrial redox state was not positively correlated with the external phosphorylation potential ([ATP]/[ADP] × [ Pi]). The following conclusions are drawn on the basis of the present data, together with other reports (Davis, Bremer, and Åkerman (1980) J. Biol. Chem. 255, 2277–2283) and (Klingenberg and Rottenberg (1977) Eur. J. Biochem. 73, 125–130) : (a) the gradient in reduction potential is driven by the membrane potential (Δψ), mediated by the electrogenic glutamate-aspartate exchange, and the poise or set point of this gradient is a function of Δψ; and (b) the gradient of ATP ADP ratios across the membrane is also driven principally by Δψ, mediated by the electrogenic ATP-ADP exchange. Hence, segregation of phosphorylation and reduction potentials is linked through a mutually shared electrical driving force.

Effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase

The effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase has been investigated. Citrate showed modest effect on the ATP-orthophosphate and ATP-ADP exchange reactions. In contrast, this tricarboxylic acid caused marked concentration-dependent stimulation of the acetyl CoA-malonyl CoA exchange reaction which was concomitant with the activation of acetyl CoA carboxylation. The data obtained are consistent with the suggestion that activation by citrate of the overall forward reaction (malonyl CoA synthesis) primarily reflects enhancement of the carboxyltransferase half-reaction catalyzed by rat mammary gland acetyl CoA carboxylase.

Cholesterol as activator of ADP-ATP exchange in reconstituted liposomes and in mitochondria

The influence of cholesterol on ADP-ATP exchange activity was measured in the reconstituted system, submitochondrial (sonic) particles and mitoplasts (isolated inner mitochondrial membranes). In the reconstituted system, cholesterol markedly enhanced the nucleotide-uptake rate, when added to membranes of various compositions i.e., pure phosphatidylcholine, phosphatidylcholine/phosphatidylethanolamine mixtures and crude egg yolk phospholipids. The stimulation was linearly dependent on the amount of incorporated cholesterol up to 7–13% added sterol, depending on the type of phospholipids. Cholesterol influenced neither the amount of actively reconstituted carrier proteins nor the affinity of the carrier towards nucleotides nor the breakpoint of temperature dependence in the Arrhenius plot. The stimulation could be correlated with an increase in the molecular activity of the carrier protein. The influence of cholesterol was also measured in the natural environment of the carrier protien, i.e., the inner mitochondrial membrane. Both with submitochondrial particles from beef heart and especially with mitoplasts from rat liver, incorporation of cholesterol by fusion with sterol-containing liposomes led to a stimulation of ADP-ATP exchange activity, comparable to the effect in the reconstituted system. These results are discussed in relation to the absence of cholesterol in the inner mitochondrial membrane and in the view of the generally accepted ordering effect of cholesterol on phospholipid bilayers.

Rhodopsin-to-metarhodopsin II transition triggers amplified changes in cytosol ATP and ADP in intact retinal rod outer segments.

We have observed rapid, light-initiated changes in unbound cytosol ATP and ADP during the rhodopsin-to-metarhodopsin II transition in intact rod outer segments (ROS). Upon illumination of the ROS, ATP is rapidly removed from the unbound phase of ROS, accompanied by the concomitant release of ADP into the cytosol. The exchange process involves decreases of approximately equal to 0.5 mM ATP in ROS cytosol ATP content in response to a saturating flash. At levels of light well below saturation (less than 0.001% bleach), the process is highly amplified, with a decrease in cytosol ATP of approximately equal to 2,000 ATP molecules per absorbed photon per ROS. Rapid time-resolution techniques reveal that cytosol ATP content decreases rapidly, within 250 msec of a saturating flash. Bleaching rhodopsin to metarhodopsin II results in a decrease in cytosol ATP, accompanied by an increase in cytosol ADP, whereas photoreversal of metarhodopsin II by a blue flash reverses the process, increasing ATP concentration to its control level in the dark. The photoreversibility of the ATP decrease during the rhodopsin-to-metarhodopsin II transition establishes a direct link between the state of an early intermediate of photolyzed rhodopsin and the state of a nucleoside triphosphate in intact ROS. The results are consistent with a light-activated exchange of unbound ATP for bound ADP, and we propose, therefore, an ATP/ADP amplification cycle in which metarhodopsin II catalyzes the exchange of ATP for ADP on a nucleotide binding protein.

Reaction of malate thiokinase with methoxycarbonyl-CoA disulfide. Evidence for half-of-the-sites reactivity.

Reaction of the active site-directed sulfhydryl reagent methoxycarbonyl-CoA disulfide with malate thiokinase follows pseudo-first order kinetics and leads to a complete loss in enzyme activity as measured by the overall reaction. The rate of inactivation of enzyme in buffer alone shows a linear dependence on the methoxycarbonyl-CoA disulfide concentration. In the presence of the substrate phosphate or the substrate analog sulfate, the rate of enzyme inactivation displays a hyperbolic response with respect to methoxycarbonyl-CoA disulfide. Anions such as chloride and fluoride decrease the rate of enzyme inactivation. Low concentrations of succinyl-CoA protect the enzyme against inactivation, while L-malate protection of the enzyme is observed only in the absence of added sulfate. Protection of the enzyme by ATP can be attributed to phosphorylation of the enzyme and a decrease in reactivity of the phosphoenzyme relative to the native enzyme. Enzyme reacted with excess methoxycarbonyl-CoA disulfide, although inactive with respect to turnover, catalyzes ATP-ADP exchange at one-half the rate of the native enzyme. The inactive enzyme can be phosphorylated by ATP, but only to 50% the extent of native enzyme. Titration of the enzyme with methoxycarbonyl-CoA disulfide shows that the enzyme exhibits all-of-the-sites reactivity with respect to this reagent. A correlation of titration of the enzyme with methoxycarbonyl-CoA disulfide with the loss in phosphorylation site shows that reaction of methoxycarbonyl-CoA disulfide produces half-of-the-sites reactivity with respect to enzyme phosphorylation.

Ca 2+-dependent activities of (Na + + K +)-ATPase

Experiments on the effects of varying concentrations of Ca 2+ on the Mg 2+ + Na +-dependent ATPase activity of a highly purified preparation of dog kidney (Na + + K +)-ATPase showed that Ca 2+ was a partial inhibitor of this activity. When Ca 2+ was added to the reaction mixture instead of Mg 2+, there was a ouabain-sensitive Ca 2+ + Na +-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg 2+ + Na +-dependent activity. The apparent affinities of the enzyme for Ca 2+ and CaATP seemed to be higher than those for Mg 2+ and MgATP. Addition of K +, along with Ca 2+ and Na +, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca 2+ + Na + + K +-dependent ATPase was about two orders of magnitude smaller than that of Mg 2+ + Na + + K +-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca 2+, Na +, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K +. The enzyme also exhibited a Ca 2+ + Na +-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca 2+ on Mg 2+-dependent activities, nor the Ca 2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg 2+ in all reaction mixtures, it could not be determined whether the apparent Ca 2+-dependent activities involved enzyme-substrate complexes containing Ca 2+ as the divalent cation or both Ca 2+ and Mg 2+.

Adenine nucleotide and lysine transport in Chlamydia psittaci

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.

Electrophoretic control of reconstituted adenine nucleotide translocation.

The initial velocity of adenine nucleotide exchange catalyzed by the reconstituted ADP-ATP carrier from beef heart mitochondria was measured under the influence of membrane potential and with different nucleotide distributions between the internal liposomal and the external buffer volume. Both Vmax and Km of adenine nucleotide uptake not only changed due to the applied potential but also depended on the respective nucleotide distribution. The rate equations for the ADP-ATP exchange under the various conditions were derived. These equations were simplified by assuming two alternative situations; either (a) af affinity type model, where the membrane potential influences only the affinity of the adenine nucleotide carrier toward ATP and ADP, or (b) a velocity type or distribution model, where the membrane potential modulates the rate constants of the ADP-ATP exchange. On the basis of several simplifications in the reconstituted system, the rate equations could be solved and the rate constants and dissociation constants of the exchange in the "energized" and in the "deenergized" state could be calculated. These values were used to derive prediction tables for normalized exchange rates under different nucleotide distributions, which were then compared with the experimental data. Only the exchange rates predicted by the velocity-type model agreed with the measured values. On the basis of this model a definite asymmetry caused by the membrane potential could be seen. Whereas this asymmetry is not very pronounced in the case of carrier-ADP complexes, about 40 times more ATP-loaded binding sites face the outside of the vesicles in the energized state.

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator.

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator control the export of ATP formed by oxidative phosphorylation in mitochondria.