ATP Exchange Reaction Research Articles

What Do Magnetic Resonance–Based Measurements of Pi→ATP Flux Tell Us About Skeletal Muscle Metabolism?

Magnetic resonance spectroscopy (MRS) methods offer a potentially valuable window into cellular metabolism. Measurement of flux between inorganic phosphate (Pi) and ATP using 31P MRS magnetization transfer has been used in resting muscle to assess what is claimed to be mitochondrial ATP synthesis and has been particularly popular in the study of insulin effects and insulin resistance. However, the measured Pi→ATP flux in resting skeletal muscle is far higher than the true rate of oxidative ATP synthesis, being dominated by a glycolytically mediated Pi↔ATP exchange reaction that is unrelated to mitochondrial function. Furthermore, even if measured accurately, the ATP production rate in resting muscle has no simple relationship to mitochondrial capacity as measured either ex vivo or in vivo. We summarize the published measurements of Pi→ATP flux, concentrating on work relevant to diabetes and insulin, relate it to current understanding of the physiology of mitochondrial ATP synthesis and glycolytic Pi↔ATP exchange, and discuss some possible implications of recently reported correlations between Pi→ATP flux and other physiological measures.

HMSH4-hMSH5 Adenosine Nucleotide Processing and Interactions with Homologous Recombination Machinery

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.

HypF, a Carbamoyl Phosphate-converting Enzyme Involved in [NiFe] Hydrogenase Maturation

HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans.

The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur J. Biochem. 267, 993-1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi <--> ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal delta pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed.

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the 32Pi–ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F 1F o ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F 1; (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Properties of bound inorganic phosphate on bovine mitochondrial F1F0-ATP synthase.

Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi <--> ATP exchange reaction with bound Pi. The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi <--> ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers.

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-->E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s-1.

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.

Aminoacyl-tRNA synthetases catalyze AMP----ADP----ATP exchange reactions, indicating labile covalent enzyme-amino-acid intermediates.

Aminoacyl-tRNA synthetases (amino acid-tRNA ligases, EC 6.1.1.-) catalyze the aminoacylation of specific amino acids onto their cognate tRNAs with extraordinary accuracy. Recent reports, however, indicate that this class of enzymes may play other roles in cellular metabolism. Several aminoacyl-tRNA synthetases are herein shown to catalyze the AMP----ADP and ADP----ATP exchange reactions (in the absence of tRNAs) by utilizing a transfer of the gamma-phosphate of ATP to reactive AMP and ADP intermediates that are probably the mixed anhydrides of the nucleotide and the corresponding amino acid. AMP and ADP produce active intermediates with amino acids by entering the back-reaction of amino acid activation, reacting with labile covalent amino acid-enzyme intermediates. Gramicidin synthetases 1 and 2, which are known to activate certain amino acids through the formation of intermediate thiol-esters of the amino acids and the enzymes, catalyze the same set of reactions with similar characteristics. Several lines of evidence suggest that these activities are an inherent part of the enzymatic reactions catalyzed by the aminoacyl-tRNA synthetases and gramicidin synthetases and are not due to impurities of adenylate kinase, NDP kinase, or low levels of tRNAs bound to the enzymes. The covalent amino acid-enzyme adducts are likely intermediates in the aminoacylation of their cognate tRNAs. The use of gramicidin synthetases has thus helped to illuminate mechanistic details of amino acid activation catalyzed by the aminoacyl-tRNA synthetases.

P i∠ATP exchange in the absence of proton gradient by the H + -ATPase from yeast plasma membranes

Purified soluble H + -ATPase from Schizosaccharomyces pombe catalyzes a P i∠ATP exchange in the absence of a H + gradient. When the pH of the assay medium is raised from 5.5 to 8.0 there is a decrease of the ATPase activity and an increase of the rate of P i∠ATP exchange. At pH 7.0 the addition of the organic solvent dimethyl sulfoxide (20%, v v ) promotes a decrease of ATPase activity and an increase of the P i∠ATP exchange reaction. The effect of the organic solvent on the P i∠ATP exchange is related to a decrease of the apparent K m for P i.

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.

Control of beef heart submitochondrial particle-catalyzed Pi goes to and comes from ATP exchange by nucleotides and the ATPase inhibitor protein.

F1I, the specific ATPase inhibitor protein, and the chromium(III) analogs of ATP and ADP, CrATP and CrADP, were used to study the inhibition of Pi goes to and comes from ATP exchange reaction catalyzed by beef heart submitochondrial particles. F1I was found to be an uncompetitive inhibitor of the exchange reaction. CrATP and CrADP, both competitive inhibitors of ATP hydrolysis in isolated F1 (Schuster, S. M., Ebel, R. E., and Lardy, H. A. (1975) ARch. Biochem. Biophys. 171, 656-661) were shown to be competitive and noncompetitive inhibitors of Pi goes to and comes from ATP exchange, respectively. Dual inhibitor studies were done using combinations of F1I and the chromium nucleotides, or the nucleotide analogs in combination. All cases show sets of intersecting Dixon plots indicative of interacting inhibitors. Upward curvature is also evident on some of the plots. This phenomenon was explained using the concept of multiple synergistic binding of the inhibitors. Binding mechanisms and their relevant kinetic equations were postulated to explain the results of the dual inhibitor studies. They support the notion that in addition to the catalytic site, there are two types of regulatory binding sites on F1, one specific for nucleotides and one specific for F1I. When one of these sites is occupied, other sites are either opened or other inhibitors become more potent.

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase.

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.

Studies on the kinetic mechanism and the phosphoryl-enzyme compound of the Escherichia coli acetate kinase reaction

The Escherichia coli acetate kinase was further examined to determine whether a phosphoryl-enzyme intermediate (or compound) was kinetically discernible or chemically essential in the catalysis of that reaction. The extent of phosphoryl-enzyme formation as monitored by gel filtration of the enzyme-phosphoryl donor substrate complex (E-P) was found to be quite variable. Isolated E-P could be reduced with sodium borohydride and the extent of formation of α-amino-δ-hydroxyvalerate paralleled the observed degree of phosphorylation of the acetate kinase. Incubation with 1 m neutral hydroxylamine of the enzyme, which had been preincubated with acetyl-P for 30–60 min, led to parallel loss of the enzyme's ability to catalyze the net reaction and the ADP ATP exchange reaction. The percentage of activity loss depended upon the concentration of acetyl-P with which the enzyme was preincubated. These observations point toward a requirement for an unmodified active site carboxyl group which may be phosphorylated during the reaction course. The theoretical basis for a new and simple alternative substrate protocol for segregating sequential and ping-pong mechanisms is presented. When applied to acetate kinase, the findings clearly rule out a ping-pong kinetic mechanism. This was also confirmed by initial rate measurements with propionyl-P and ADP, and the weight of the evidence now clearly favors a modified random substrate addition pathway as presented herein. The data do not in any way exclude E-P formation as a requirement for catalysis. Finally, the rates of cold inactivation at 0 °C and reactivation at 29 °C were determined in order to minimize the possible influence of this phenomenon on the kinetic studies.

Dependency of the ATPase and 32P—ATP exchange reaction of mitochondria on K + and electron transport

The 2,4-dinitrophenol-stimulated ATPase activity and the 32P-ATP exchange reaction has been studied in rat liver mitochondria having less than 15 nmoles of K + per milligram of protein. With 200 m m sucrose in the incubation media, the permeation of K + and an oxidizable substrate is required for maximal stimulation of ATPase activity by 2,4-dinitrophenol. In these conditions, the 2,4-dinitrophenol-stimulated ATPase is inhibited by antimycin, acetate and mersalyl and depends to a certain extent on the rate of electron transport. The 32P-ATP exchange reaction of mitochondria with a low content of K + also requires K + permeation and is inhibited by antimycin, cyanide, 2,4-dinitrophenol, and acetate. The results suggest that the entrance of ATP into the mitochondria is compulsory linked to K + uptake in a process that depends on a negative internal potential.

Promitochondria of anaerobically grown yeast. V. Energy transfer in the absence of an electron transfer chain.

Promitochondria of anaerobically grown Saccharomyces cerevisiae lack cytochromes aa(3), b, c(1), and c, as well as ubiquinone, yet catalyze a P(i)-ATP exchange reaction that is sensitive to uncouplers, rutamycin, and atractyloside. The promitochondrial P(i)-ATP exchange reaction is abolished by the cytoplasmic "petite" mutation, as well as by growth of the cells in the presence of erythromycin, which indicates a role of mitochondrial protein synthesis in the assembly of the energy transfer system. These observations demonstrate that mitochondrial energy transfer can occur in the absence of a respiratory chain.

Effects of cystamine and cysteamine on the adenosine-triphosphatase activity and oxidative phosphorylation of rat-liver mitochondria.

1. Cystamine (2,2'-diaminodiethyl disulphide) caused an unmasking of mitochondrial adenosine triphosphatase and a leakage of Mg(2+) from the mitochondria, and decreased the stimulation of adenosine triphosphatase by 2,4-dinitrophenol. When Mg(2+) was added, cystamine potentiated the activation of adenosine triphosphatase by 2,4-dinitrophenol. 2. Cystamine was without effect on the adenosine triphosphatase of disrupted mitochondria. 3. Cystamine was moderately potent as an uncoupling agent and as an inhibitor of the [(32)P]P(i)-ATP exchange reaction. 4. Cysteamine (2-aminoethanethiol) was without the above effects, when special precautions were taken to counteract its autoxidation. 5. The effects of cystamine should probably be ascribed to its disulphide group, since the diamine cadaverine protected slightly against the loss of Mg(2+) and the decrease of 2,4-dinitrophenol-stimulated adenosine-triphosphatase activity caused by aging of the mitochondria. It is suggested that cystamine acts by a breakdown of mitochondrial permeability barriers.

Activity of Amino Acid-activating Enzymes in Tissues from Protein-depleted Rats

The activity of amino acid-activating enzymes in tissues from protein-depleted rats was tested by the PPi - P32 ⇄ ATP exchange reaction. Previously we had found that following protein depletion and in concomitance with a greater incorporation of amino acids, the activity of the liver amino acid-activating enzymes significantly increased. Presently the aim was to test the possible generalization of the above by investigating whether the activity of the amino acid-activating enzymes in various tissues of protein-depleted rats would vary in a manner analogous to the differences reported for amino acid incorporation in these tissues. The results, besides confirming that in protein depletion the liver amino acid-activating enzymes increase when tested in the presence of the complete mixture of amino acids or in the presence of individual amino acids, have also shown that in heart and gastrocnemius muscle the level of these enzymes is less than normal, in agreement with the decreased amino acid incorporation in these organs. A general hypothesis was thus set up that, in tissues from protein-depleted animals, the amount of protein synthesis is regulated through modifications of the level of amino acid-activating enzymes.