ADP-ATP Exchange Research Articles

Leucine 332 at the boundary between the fourth transmembrane segment and the cytoplasmic domain of Na+,K+-ATPase plays a pivotal role in the ion translocating conformational changes.

Mutants Gly330-->Ala, Leu332-->Ala, Leu332-->Pro, and Pro780-->Ala of the alpha1-isoform of rat kidney Na+,K+-ATPase were expressed in COS-1 cells to active site concentrations between 20 and 70 pmol per mg of membrane protein. The functional properties of the mutants were characterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-ATPase activity as well as by a series of assays measuring the K+-dependence of the steady-state phosphoenzyme level, the kinetics of dephosphorylation under a variety of conditions, and the ADP-ATP exchange activity. In mutants Gly330-->Ala, Leu332-->Ala, and Leu332-->Pro, the molecular turnover number was reduced relative to that of the wild-type Na+,K+-ATPase, and the steady-state phosphoenzyme level was high even in the presence of several millimolar K+. At a low Na+ concentration in the absence of K+, mutants Leu332-->Pro and Gly330-->Ala displayed high ADP-ATP exchange activity and formed a high level of the ADP-sensitive phosphoenzyme (E1P). The phosphoenzyme decayed slowly following a jump in salt concentration and chase with ATP and K+. Hence, the conversion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in mutants Leu332-->Pro and Gly330-->Ala. In the Leu332-->Ala mutant, a high level of E2P was accumulated in the absence of K+, and the ADP-ATP exchange activity was low at low Na+ concentration in the absence of K+, but rose sharply on addition of K+. Dephosphorylation experiments indicated that in the Leu332-->Ala mutant K+ induced reversal of the phosphoenzyme interconversion, forming E1P from E2P. Leu332 is therefore a pivotal residue in the conformational change. Mutants Gly330-->Ala and Pro780-->Ala displayed reduced K+ affinities relative to the wild type, determined in three independent assays.

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots.

ATP-ADP exchange was estimated in the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-100 by measuring [14C]ATP formation from [14C]ADP. Most studies were done at 0[deg]C. At pH 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 [mu]M and (b) insensitivity to ionic strength, [Na+], and [K+]. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E1ATP [left right arrow] E1P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0[deg]C the exchange showed a 3-fold pH increase, from pH 5.5 to 9.0. At an alkaline pH the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenyl-hydrazone, had a biphasic response to Mg2+ (maximal at 513 [mu]m), and was insensitive to ionic strength. At 20[deg]C ATP-ADP exchange was pH insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around pH 6.0 to 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.

Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers

The contractile properties of rabbit skinned muscle fibers were studied at 1-2 degrees C in different concentrations of MgATP and MgADP. Double-reciprocal plots of maximum velocity against MgATP concentration at different MgADP concentrations all extrapolated to the same value. This finding suggests that MgATP and MgADP compete for the same site on the cross-bridge, and that the exchange of MgATP for MgADP occurs without a detectable step intervening. The K(m) for ATP was 0.32 mM. The K(i) for MgADP was 0.33 mM. Control experiments suggested that the tortuosity of diffusion paths within the fibers reduced the radial diffusion coefficients for reactants about sixfold. Increasing MgADP from 0.18 to 2 mM at 5 mM ATP or lowering MgATP from 10 to 2 mM at 0.18 mM MgADP, respectively, increased isometric force by 25% and 23%, increased stiffness by 10% and 20%, and decreased maximum velocity by 35% and 31%. Two mechanisms appeared to be responsible. One detained bridges in high-force states, where they recovered from a length step with a slower time course. The other increased the fraction of attached bridges without altering the kinetics of their responses, possibly by an increased activation resulting from cooperative effects of the detained, high-force bridges. The rigor bridge was more effective than the ADP-bound bridge in increasing the number of attached bridges with unaltered kinetics.

Kinetics of ATP to ADP beta-phosphoryl conversion in contracting skeletal muscle by in vivo 31P NMR magnetization transfer.

The rate constant of the beta-adenosine triphosphate to beta-adenosine diphosphate conversion was measured using 31P nuclear magnetic resonance magnetization transfer in resting and contracting in vivo rat skeletal muscle. Theoretically, the rate constant should be the sum of the rate constants of the reactions catalyzing ATP-ADP exchange. In resting muscle, the conversion rate constant was 0.4 s-1 and beta-ATP intrinsic T1 was 1.7 s. The velocity of conversion was 3.8 mM s-1. During stimulation, phosphocreatine fell to 36% and ATP to 82% of initial values. The rate constant and velocity of beta-phosphoryl conversion increased to 0.8 s-1 and 6.3 mM s-1, respectively, but did not reach expected levels, i.e. the product of the ATP concentration with the sum of pseudo first-rate constants of the individual reactions. These conversion velocities should be higher than reverse creatine kinase velocities, previously measured to be 10 mM s-1 in resting muscle and 7.5 mM s-1 in contacting muscle and confirmed in this work. The discrepancy between expected and observed data could be due either to compartmentation of part of the beta-ATP in pools exchanging slowly with the bulk of cellular ATP, or to ADP binding to macromolecules thus preventing full ADP saturation during magnetization transfer.

The speed of partial reactions of the uncoating ATPase Hsc70 depends on the source of coated vesicles.

Hsc70 was previously isolated by its ability to catalyse the uncoating of clathrin-coated vesicles from bovine brain. We have recently shown that Hsc70 is more active towards coated vesicles from brain than those from other tissues. In order to gain information on the mechanistic reason for this difference we have examined the ability of brain and placental coated vesicles to stimulate partial reactions during a single round of ATP turnover. The Hsc70-ATP complex is turned over to Hsc70-ADP center dot Pi, from which phosphate is slowly released. The resulting Hsc70-ADP complex exchanges ATP for ADP. Dissociation of ATP or ADP from Hsc70 does not seem to occur under physiological conditions. The hydrolysis of ATP is accelerated by the presence of clathrin-coated vesicles, with vesicles from brain being about twice as effective as vesicles from placenta. Additionally, it appears that brain, but not placental, coated vesicles can also stimulate the exchange of ADP for ATP.

Mechanism for the Coupling of ATP Hydrolysis to the Conversion of 5-Formyltetrahydrofolate to 5,10-Methenyltetrahydrofolate

5,10-Methenyltetrahydrofolate synthetase catalyzes the irreversible conversion of 5-formyl-tetrahydropteroylpolyglutamates (5-CHO-H4PteGlu(n)) to 5,10-methenyltetrahydropteroylpolyglutamates (5, 10-CH(+)-H4PteGlu(n)). The equilibrium of the nonenzymatic reaction, which equilibrates slowly in the absence of enzyme, greatly favors 5-CHO-H4PteGlu(n). The enzyme couples the reaction to the hydrolysis of ATP shifting the equilibrium to favor 5,10-CH(+)-H4PteGlu(n). Substrate-dependent non-equilibrium isotope exchange of [3H]ADP into ATP was observed, suggesting the formation of a phosphorylated intermediate of 5-CHO-H4PteGlu(n) during the enzyme-catalyzed reaction. The competitive inhibitor 5-formyltetrahydrohomofolate also supported the ADP to ATP exchange, suggesting that this molecule could also form a phosphorylated intermediate. The initial rates of the ADP-ATP exchange with saturating ADP were about 70 s-1 for both compounds, while the kcat values for product formation were 5 s-1 for 5-CHO-H4PteGlu(n) and 0.005 s-1 for 5-formyltetrahydrohomofolate. Starting with 5(-)[18O]CHO-H4PteGlu(n), it was shown by 31P NMR that the formyl oxygen of the substrate was transferred to the product phosphate during the reaction. This further supports the existence of a phosphorylated intermediate. The formyl group of 5-CHO-H4PteGlu(n) is known to be an equilibrium mixture of two rotamers. Stopped-flow analysis of the enzymatic reaction showed that only one of the rotamers serves as a substrate for the enzyme.

The chaperonin cycle and protein folding

The process of protein folding in the cell is now known to depend on the action of other proteins. These proteins include molecular chaperones, which interact non-covalently with proteins as they fold and improve the final yields of active protein in the cell. The precise mechanism by which molecular chaperones act is obscure. Experiments reported recently show that for one molecular chaperone (Cpn60, typified by the E. coli protein GroEL), the folding reaction is driven by cycles of binding and release of the co-chaperone Cpn10 (known as GroES in E. coli). These alternate with binding and release of the unfolded protein substrate. These cycles come about because of the opposite effects of Cpn10 and unfolded protein on the Cpn60 complex: the former stabilises the ADP-bound state of Cpn60, whereas the latter stimulates ADP-ATP exchange. This model proposes that the substrate protein goes through multiple cycles of binding and release, and is released into the cavity of the Cpn60 complex where it can undergo folding without interacting with other nearby folding intermediates. This is consistent with the ability of Cpn60 proteins to enhance folding by blocking pathways to aggregation.

Examination of catalytic activity of the β subunit isolated from chloroplast coupling factor 1

A simple and rapid procedure has been developed to isolate the subunit from chloroplast coupling factor 1 involving anion exchange HPLC. The β subunit was mainly in the monomeric form, along with some quantities of trimer and more complex aggregates of the protein. Decreasing pH down to neutral values or heating resulted in shifting the equilibrium in the medium to the formation of polymeric forms of the β subunit. By successive purification and fractionation of CF 1 and β subunit preparations, it was shown that the monomeric form of the β subunit catalysed ATP-ADP γ-phosphate exchange. However, it did not display ATPase or adenylate kinase activities. Aggregation of the β subunit was accompanied by irreversible inactivation of its ATP-ADP exchange activity.

The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding.

The reaction mechanism of protein folding by the chaperonin GroEL and its regulator GroES has been defined. GroES and substrate protein counteract each other's effects on GroEL: whereas GroES stabilizes GroEL in the ADP-bound state, binding of unfolded polypeptide within the cavity of the GroEL cylinder triggers ADP and GroES release. Upon ADP-ATP exchange, GroES reassociates with GroEL and ATP hydrolysis discharges the bound protein for folding. Partially folded protein rebinds to the chaperonin, thus perpetuating the cycle until folding is complete.

Catalytic properties of ? subunit isolated from chloroplast coupling factor 1

The chloroplast coupling factor (CF1) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the β subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The β subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg(2+)-dependent ATPase activity of CF1. However, it manifested appreciable adenylate kinase-like and ATP-ADP γ-phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg(2+) ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg(2+)-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF1-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the β subunit.

Inhibition of Ca 2+ accumulation in isolated sarcoplasmic reticulum by thyroid hormones

Thyroid hormones inhibit Ca 2+ accumulation and ATPase activity of isolated sarcoplasmic reticulum vesicles. Half-maximal inhibition was obtained by about 2.5 μM. The ATP hydrolysis activity of the purified (Ca 2+ + Mg 2+)-ATPase or the SR vesicles, in the presence of the Ca 2+ ionophore A23187, is not inhibited by T 3 or T 4. Modification of T 3 or T 4 in the ring portion, but not in the amino portion, of te molecules results in T 4 and T 3 analogues which are unable to inhibit Ca 2+ accumulation. T 3 and T 4 have no significant effect on various partial reactions of the transport cycle such as: the binding of ATP and Ca 2+, or ADP-ATP exchange and E-P formation from ATP, but they inhibit the E-P formation from inorganic phosphate (P i) and ATP-P i exchange. The inhibition of both Ca 2+ accumulation and ATPaseactivity by T 3 or T 4 is increased in the presence of P i. Binding sites for [ 125I]T 3 and for [ 125I]T 4 in SR proteins were demonstrated using either equilibrium dialysis or gel overlay techniques. The results suggest that the thyroid hormones inhibit the ATP-dependent Ca 2+ accumulation, probably by inhibiting the transport of anions which act as the Ca 2+ precipitating anion.

A reversible conformational transition in muscle actin is caused by nucleotide exchange and uncovers cysteine in position 10.

ATP-G-actin in the absence of excess ATP and divalent metal ions was treated with ADP in amounts large enough to ensure complete formation of ADP-G-actin. Under these conditions the monomer undergoes a very slow structural transition as seen by the exposure of 2.0 +/- 0.2 thiol groups per actin molecule. Once exposed, the second thiol group reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) at a rate approximately 10-fold higher than that of cysteine 374. Labeling experiments with 2,4-dinitrophenyl [1-14C]cysteinyl disulfide followed by digestion and peptide analysis showed (besides reaction with cysteine 374) nearly exclusive labeling of cysteine 10. Since this residue is completely shielded in ATP-G-actin, exchange of ATP for ADP must have caused a partial unfolding of the protein uncovering the side chain of this cysteine. The transition is reversible, because addition of ATP or of excess divalent metal ions restored the conformation with only cysteine 374 exposed. Reversibility of the transition allowed us to directly determine the relative affinities of ATP and ADP to monomeric actin in the absence of Me2+ ions. By determination of the 50% exposure value of cysteine 10 from either side of the equilibrium we found a value of KATP/KADP = 30. The rate of uncovering of the thiol of cysteine 10 at 0 degree C was distinctly slower (t1/2 = 9 h) than its reshielding by the addition of ATP (t1/2 = 3 h). The structural change was accompanied by a decrease in polymerization rate. Relative polymerization rates were determined as ATP-G(1S)-actin:ADP-G(approximately 1S)-actin:ADP-G(2S)-actin = 1.0:0.35:0.1. From the data presented here we conclude that preparations of ADP-G-actin remain undefined unless the number of thiol groups exposed has been determined.

Autoregulation of the phosphointermediate of Na +/K +-ATPase by the amino-terminal domain of the α-subunit

Chymotryptic cleavage of the α-subunit of the canine kidney Na +/K +-ATPase in the presence of Na + abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E 1 to E 2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of α is dependent on Na + and prevented by K +, that of the 83 kDa peptide is modestly stimulated by Na +; and only this stimulation, but not the Na +-independent phosphorylation is inhibited by K +. Ouabain, which inhibits α-phosphorylation by ATP, activates Na +-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na +-stimulation of this process. While there is a ouabain-stimulated phosphorylation of α by P i, the 83 kDa peptide is not phosphorylated by P i with or without ouabain. In its sensitivity to ADP, and insensitivity to K +, the phosphopeptide is similar to the E 1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na + has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na + concentrations ( K 0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na +. The Na +-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na +, K + and ouabain are autoregulated by the NH 2-terminal domain of the α-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E 1-E 2 transitions is not valid.

Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase.

We have investigated here the pre-steady state kinetics of sarcoplasmic reticulum ATPase incubated under conditions where significant amounts of Mg.ATP and Ca.ATP coexist, both of them being substrates for the ATPase. We confirmed that these two substrates are independently hydrolyzed by the ATPase, which thus apparently catalyzes Pi production by two simultaneous and separate pathways. External calcium (or the Ca2+/Mg2+ ratio) determines the extent to which Ca2+ or Mg2+ is bound at the phosphorylation site, while internal calcium controls the rate of processing of both the slow, calcium-containing and the fast, magnesium-containing phosphoenzyme. Time-dependent binding of calcium at the catalytic site is correlated with the observed burst of Pi liberation, which therefore results from reequilibration during pre-steady state of magnesium- and calcium-containing phosphoenzyme pools. Independently of direct exchange of metal at the catalytic site, ADP produced by the hydrolysis reaction contributes to reequilibration of these pools through reversal of phosphorylation by the ATP-ADP exchange pathway.

Distinction between the intermediates in Na +-ATPase and Na +,K +-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations

The ATP hydrolysis rate and the ADP-ATP exchange rate of ( Na + + K +)- ATPase from ox brain were measured at 10 μM Mg free 2+ and at micromolar concentrations of free ATP and ADP. (1) In the absence of K +, substrate inhibition of the hydrolysis rate was observed. It disappeared at low Na + and diminished at increasing concentrations of ADP. This was interpreted in terms of free ATP binding to E 1P. In support of this interpretation, free ATP was found to competitively inhibit ADP-ATP exchange. (2) In the presence of K +, substrate activation of the hydrolysis rate was observed. Increasing (μM) concentrations of ADP did not give rise to competitive inhibition in contrast to the situation in the absence of K + (cf. 1, above). This was interpreted to show that at micromolar substrate, some low-affinity, high-turnover Na + + K + activity is possible, provided the Mg 2+ concentration is low. (3) While small concentrations of K + increased the hydrolysis rate (cf. 2) they decreased the rate of ADP-ATP exchange. To elucidate this phenomenon, parallel measurements of exchange and hydrolysis rates were performed over a wide range of ATP and ADP concentrations, with and without K +. If, in the presence and absence of K +, ADP (and ATP competing) are binding to the same phosphorylated intermediate for the backward reaction, it places quantitative restrictions on the ratio of rate constants with and without K +. The results did not conform to these restrictions, and the discrepancy is taken as evidence for the necessity for a bicyclic scheme for the action of the ( Na + + K +)- ATPase . (4) An earlier statement concerning the nature of the phosphoenzyme obtained in the presence of Na + and K + is amended.

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP.

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.

Fast reversal of the initial reaction steps of the plasma membrane (Ca 2+ + Mg 2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca 2+, Mg 2+-dependent ATP-[ 3H]ADP exchange at 37° C. The Ca 2+, Mg 2+-dependent exchange, measured at 20 μM CaCl 2, 1.5 mM MgCl 2, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca 2+ + Mg 2+)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca 2+-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg 2+-containing medium, while their Ca 2+-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl 2 to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca 2+-dependent ATP-ADP exchange, slightly less than it stimulates the Ca 2+-dependent ATP hydrolysis. The effect of 1.5 mM MgCl 2 on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca 2+ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca 2+ pump should then be due to one or more reaction steps later than and including Ca 2+ release and dephosphorylation.

Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.

Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K-ATPase.

Interactions of Na, K, Ca and Mg ions with partially purified Na,K-ATPase from pig kidneys were investigated using as a tool simultaneous determinations of adenosine 5'-triphosphate (ATP)-adenosine 5'-diphosphate (ADP) exchange and ATPase activity catalysed by the enzyme. In the presence of 120 mM-NaCl and 0.5 mM-MgCl2, the effects 12 mM-KCl on ATP-ADP exchange depended on the ATP and ADP concentrations: stimulation (about 10-fold) with 3 mM-ATP-0.75 mM-ADP, no effect with 0.04 mM-ATP-0.01 mM-ADP and inhibition when ATP and ADP concentrations were 0.003 mM and 0.001 mM respectively. The apparent affinities (K0.5) for K stimulation of ATP-ADP exchange and Na,K-ATPase activity were indistinguishable from each other both at 20 mM- and 120 mM-Na. All Na and Na+K-dependent exchanges were completely abolished by 10(-4) M-ouabain. In the absence of K, intermediate Na concentrations were inhibitory of ouabain-sensitive ATP-ADP exchange and ATPase activity. This Na inhibition was more pronounced at low than at high ionic strength and was more noticeable in the exchange reaction. K ions antagonized these Na effects; this K-Na antagonism was more effective on ATPase than on the ATP-ADP exchange. Ca ions, in the absence of Mg, could sustain ATP-ADP exchange. Qualitatively the behaviour of the exchange reaction was similar to that observed with Mg. Quantitatively the rates were always lower with Ca. In the presence of Mg, at low ionic strength and at concentrations which stimulated phosphatase activity, Ca ions were not able to counteract Na inhibition of ATPase and ATP-ADP exchange or to stimulate ATPase activity in the presence of Na. This is another indication of the different reactivity of the K sites involved in phosphatase and ATPase activities as well as of those responsible for the release of Na inhibition of ATPase and exchange reaction. In K-free solutions containing 0.5 mM-Na and low ionic strength, Mg was a powerful inhibitor of ouabain-sensitive ATPase and ATP-ADP exchange; the curves relating remaining activities to Mg concentrations were identical for both reactions. When Na was increased to 5 mM, ATP-ADP exchange became more sensitive and ATPase more resistant to Mg inhibition. These data are consistent with (i) a Na-Mg antagonism at the phosphorylation step, and (ii) a Na-Mg synergism in blocking the E2PNa-E1PNa transformation.(ABSTRACT TRUNCATED AT 400 WORDS)

The dependence on internal pH of Ca2+ -fluxes across sarcoplasmic reticulum vesicular membranes

The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low-affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH-dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH-jump method) were observed. In addition, Cai-Cao exchange in the absence and presence of ADP and ATP-ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high-affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations. Active Ca2+ transport (particularly evident applying the pH-jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH-dependence of passive Ca2+ accumulation and the initial rate of Cai-Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai-Cao exchange, and the ATP-ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low-affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.