Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-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-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.     

Noncooperative stabilization effect of phalloidin on ADP.BeFx- and ADP.AlF4-actin filaments

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.     

Inhibition by ADP of prostaglandin induced accumulation of cyclic AMP in intact human platelets

The influence of extracellular ADP on cyclic AMP accumulation within intact human platelets was studied. ADP inhibited the increase in cyclic AMP which occurs when platelets are exposed to prostaglandin E1 or I2. The degree of inhibition varied in the range 70-95% , and was half maximal at ADP concentrations of between 0.3 and 2 microM. Other naturally occurring diphosphates, i.e. GDP, IDP and UDP, were at least 100 fold less effective than ADP, and UDP at 1mM partially reversed the effect of ADP. The effect by ADP was completely reversed by ATP, but only attenuated to a minor degree of 10 mM EDTA. Increasing concentrations of ADP caused a progressive degree of inhibition of cyclic AMP accumulation, and the kinetics of this inhibition were compatible with a simple saturable process with no cooperativity. ADP added 10 seconds after prostaglandin E1 blocked cyclic AMP accumulation within 1-2 seconds, and addition of ATP after ADP and prostaglandin I2 relieved the inhibition due to ADP within 2-3 seconds. The action of ADP was blocked by sulphydryl reagents including N-substituted maleimides, cytochalasin A, NBD chloride and p-mercuribenzene sulphonate. The data were considered to be consistent with mediation of the ADP effect through a sulphydryl-bearing specific extracellular receptor coupled to the adenylate cyclase.     

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory role of phosphate and other anions in transport of ADP and ATP by Rickettsia prowazekii.

ADP and ATP were transported in Rickettsia prowazekii by an obligate exchange system without prior hydrolysis. The uptake of ATP and ADP by the obligate exchange system in R. prowazekii was dependent upon the anionic composition of the medium. The rate of transport of ATP was about three times greater than that of ADP in the absence of anions, and the rates of transport of both were about doubled by a variety of anions. However, phosphate anions were able to stimulate greatly the uptake of ADP so that in the presence of these anions, the uptake of ATP and that of ADP were about equal. Millimolar concentrations of anions were required to elicit the stimulation of ADP and ATP transport. The ADP-dependent efflux of ADP and ATP was also greatly stimulated by phosphate anions. The stimulation of ADP and ATP transport required that the anions be present in the external medium, as preincubation of the rickettsiae with phosphate anions was neither necessary nor sufficient. The competitive inhibition of ATP uptake by ADP required phosphate anions, indicating that phosphate anions increased the affinity of ADP for the transport system. The role of phosphate in the regulation of ATP and ADP exchange and its significance are discussed.     

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.     

An improved method for the isolation of dense storage granules from human platelets

Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase

The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.     

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.     

Nucleotide exchange in membrane vesicles from the photosynthetic bacterium Rhodopseudomonas capsulata

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [³H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 mm. The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [³H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [³H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.     

Initial events of light-dependent ATP synthesis in spinach subchloroplast particles.

The kinetics of 32Pi incorporation into adenine nucleotides by subchloroplast particles in the light is studied with a continuous flow apparatus allowing measurements between 3 and 200 ms. After a short lag time from 1 to 3 ms ATP synthesis proceeds with a constant rate. During the first few milliseconds a faster labelling of ADP is detected. This labelling of ADP reaches a constant level up to 1 molecule ADP labelled per molecule of coupling factor present. The labelling pattern in ATP indicates that the labelled ADP does not equilibrate with free ADP. The addition of 32Pi to a phosphorylating system during the light phase (32Pi pulse) exhibits unchanged kinetic characteristics for labelling of ATP and ADP. These results indicate a phosphorylation of AMP to ADP being an intermediate step in photophosphorylation. In experiments carried out in the dark no label is found in ATP within the time analysed. However the labelling of ADP occurs in the same way as in the light.     

A fluorometric study with 1-anilinonaphthalene-8-sulfonic acid (ANS) of the interactions of ATP and ADP with rubisco activase

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to the protein. Evidence of conformational changes was observed from the differences in the interaction of ANS with rubisco activase in the presence of excess ATP and ADP. Fluorescent changes associated with the titration of a rubisco activase-ANS mixture with ATP and ADP indicated that the binding of ADP to rubisco activase was much tighter than that of ATP. The concentration of Mg2+ and pH had significant effects on the affinities of rubisco activase for ATP and ADP, with higher pH and Mg2+ concentration facilitating the binding of ATP to rubisco activase in the presence of ADP. The physiological implications of the binding characteristics of ATP and ADP with rubisco activase on the light-dark regulation of rubisco are discussed.     

Regulation of Ca2+ transport in brain mitochondria. II. The mechanism of the adenine nucleotides enhancement of Ca2+ uptake and retention

ADP greatly enhances the rate of Ca2+ uptake and retention in Ca2+ loaded mitochondria. Atractyloside, a specific inhibitor of the ADP/ATP translocator, completely inhibits the ADP effect, while bongkrekate, another specific inhibitor of the translocator enhances the effect of ADP. These results indicate that locking the ADP/ATP translocator in the M-state is sufficient to produce the ADP effect. Cyclosporin A, a specific inhibitor of the Ca2(+)-induced membrane permeabilization does not substitute for ADP, indicating that ADP directly affect the rate of electrogenic Ca2+ uptake. The effect of the translocator conformation on the rate of electrogenic Ca2+ uptake is independent of the concentration of Pi and is not caused by changes in membrane potential. However, locking the carrier in the M-state appears to increase the negative surface charge on the matrix face of the inner membrane. This may lead to an enhanced rate of Ca2+ dissociation from the electrogenic carrier at the matrix surface. The rate of Na(+)-independent Ca2+ efflux is only slightly inhibited by locking the carrier in the M-state, presumably due to the same mechanism. In the presence of ADP, Pi inhibits the Na(+)-independent efflux. In the presence of physiological concentrations of spermine, Pi and Mg2+, the rate of Ca2+ uptake, Ca2+ retention and Ca2+ set points depend sharply on ADP concentration at the physiological range of ADP. Thus, changes of cytosolic ADP concentration may lead to change in the rate of Ca2+ uptake by mitochondria and thus modulate the excitation-relaxation cycles of cytoplasmic free calcium.     

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.     

Regulation of glycolysis by adenosine diphosphate in Pisum sativum

The role of ADP in controlling glycolysis has been examined in a soluble extract of germinating pea seeds. A shortage of ADP appears to retard glycolysis principally by restricting the conversion of phosphopyruvate to pyruvate rather than by restricting formation of phosphoglycerate. Upon addition of ADP to the extract there is an immediate decrease in the concentration of phosphopyruvate accompanied by an increase in pyruvate. Apparently the pyruvate-kinase step shows the most marked response to fluctuations in ADP availability. The glycolytic response to ADP depends on the concentration of ATP magnesium ions. The relation of magnesium-ion availability to adenine-nucleotide control of glycolysis is discussed.     

Tentoxin-induced binding of adenine nucleotides to soluble spinach chloroplast coupling factor 1.

The effect of tentoxin on the binding of adenine nucleotides to soluble chloroplast coupling factor (CF1) has been studied and the following results have been obtained: 1. Tentoxin (400 micron) increases the maximum attainable tight binding of ADP to CF1. In the absence of tentoxin, the maximal binding observed by the method employed is about 0.3 nmol ADP/mg protein, whereas in the presence of tentoxin this ranges from 1.5 to 2.0 nmol ADP/mg protein. 2. Tentoxin-induced binding of ADP to CF1 is severely inhibited by divalent cations (50% inhibition at about 2 mM) but only weakly inhibited by monovalent cations (less than 50% inhibition at 100 mM). 3. The binding of ADP to CF1 induced by tentoxin is inhibited by ATP and adenylyl imidodiphosphate but is not inhibited by other nucleotides including AMP, GDP, CDP, IDP, or beta, gamma-methylene ATP. 4. The ADP-CF1 complex induced by tentoxin is quite stable. 75% remains bound to CF1 even after passage of the complex through a gel filtration column. An additional 25% can be removed by incubation in the presence of ADP, and all of the bound ADP can be removed only after incubation in the presence of both tentoxin and ADP. The latter result is interpreted as a tentoxin-induced exchange of bound ADP for medium ADP.     

On the interrelationship of prostaglandin endoperoxide G2 and cyclic nucleotides in platelet function.

The prostaglandin endoperoxide G2 caused rapid aggregation and relase of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate/creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide. The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3':5'-monophosphate (cAMP) levels in platelet-rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet-rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2-Dibutyrylguanosine 3':5'-monophosphate did not influence the aggregation and release-reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.     

Use of helper enzymes for ADP removal in infrared spectroscopic experiments: application to Ca2+-ATPase

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca(2+)-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free P(i), which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca(2)E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca(2)E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.     

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.