Inhibition of IMP-specific cytosolic 5''-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5''-deoxy-5''-isobutylthio derivatives of adenosine and inosine.

1. The partially purified IMP-specific cytosolic 5'-nucleotidases from rat liver, polymorphonuclear leucocytes and heart were inhibited by 50% by 2-6 mM-5'-deoxy-5'-isobutylthioadenosine (IBTA) or 7-10 mM-5'-deoxy-5'-isobutylthioinosine (IBTI). IBTA and IBTI inhibited the rat liver and polymorphonuclear-leucocyte enzymes non-competitively. IBTA, but not IBTI, also inhibited the ecto-5'-nucleotidase of polymorphonuclear leucocytes. IBTI was, by contrast, a more potent inhibitor than IBTA of the AMP-specific soluble 5'-nucleotidase from pigeon heart. 2. During 2-deoxyglucose-induced ATP-catabolism in rat polymorphonuclear leucocytes, adenosine formation was inhibited by approx. 80% by 3 mM-IBTA and by approx. 70% by 7 mM-IBTI. 3. The results show that 5'-modified nucleosides are inhibitors of cytosolic 5'-nucleotidases and that they penetrate to inhibit their target enzymes in intact cells. Such inhibitors may be useful to clarify the mechanisms of adenosine formation and to prevent mononucleotide hydrolysis during ATP breakdown.     

Adenosine production inside rat polymorphonuclear leucocytes.

Adenosine synthesis was studied during 2-deoxyglucose-induced ATP catabolism in intact rat polymorphonuclear leucocytes. When both adenosine kinase (EC 2.7.1.20) and adenosine deaminase (EC 3.5.4.4) were selectively inhibited, adenosine accumulated. Adenosine formation took place inside the intact cells by a metabolic pathway independent of the ecto-5'-nucleotidase (EC 3.1.3.5). Distinct metabolic pathways are proposed for adenosine production from intracellular or extracellular nucleotides.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

5''-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart.

Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum

Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

5'-Nucleotidase activity and adenosine production in rat liver mitochondria.

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.     

Adenine nucleotides in cholinergic transmission: presynaptic aspects.

1. Isolated nerve terminals (T-sacs and synaptosomes) prepared from the purely cholinergic Torpedo electric organ have been studied for their ability to incorporate and metabolise [2-3H] adenosine and to degrade 5'-AMP to adenosine. 2. A temperature-dependent, saturable uptake system for adenosine was found with kinetic properties similar to nucleoside transport systems in other cells. The uptake system in Torpedo nerve terminals was inhibited by 2'-deoxyadenosine, a known inhibitor of adenosine transport. 3. Intraterminal adenosine is rapidly metabolised to a number of products including AMP, ADP and ATP. 4. Isolated nerve terminals contain considerable 5'-nucleotidase activity, most of which resides on the outer face of the external membrane. The Km of the enzyme is congruent to 5 micron and it is inhibited by a phosphonate analogue of ADP, alpha-beta-methylene-ADP. It is suggested that this 5'-nucleotidase plays an important role in the production of adenosine from a nucleotide pool in the synaptic cleft.     

Metabolism of Extracellular Adenine Nucleotides by Cultured Rat Brain Astrocytes

Intact astrocytes cultured from newborn rat cerebral cortex rapidly converted extracellular ATP to ADP. The ATPase responsible was apparently not saturated, even at 750 microM ATP. In contrast, the conversion of ADP to AMP was slow, and the reaction was limiting for the subsequent dephosphorylation process. Adenosine formation was the only fate for AMP. The reaction was catalyzed by 5'-nucleotidase with an apparent Km of 55 microM for AMP and appeared to be inhibited by high concentrations of ATP and ADP. Astrocytes were able to take up adenosine with an apparent Km value of 45 microM. Uptake was inhibited by dipyridamole but not by anti-5'-nucleotidase IgG. The results support the proposal that astrocytes play a role in modulating synaptic events involving ATP and adenosine.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Ecto-5'-nucleotidase of cultured rat mesangial cells

Because adenosine plays a role in the regulation of glomerular filtration rate and of the release of renin, we examined the possibility of a local source for this mediator. We found that rat cultured glomerular mesangial cells converted 5'-AMP into adenosine. The properties of the enzyme involved in the reaction were those of an ecto-5' nucleotidase: (1) the products of the reaction were generated in the extracellular fluid although no 5'-nucleotidase was released by the cells into the medium; (2) identical activities were found for cultured cells in situ and sonicated cells; (3) the diazonium salt of sulfanilic acid which is a nonpenetrating reagent inhibited up to 75% of the enzyme activity. Ecto-5'-nucleotidase activity of intact cells obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 0.32 mM. 5'-UMP was a strictly competitive inhibitor. ADP exerted a very powerful inhibitory effect and behaved also as a competitive inhibitor. ATP was inhibitory both by increasing Km and by decreasing Vmax. Ecto-5'-nucleotidase was active in the absence of divalent cations. However, Mg2+, Ca2+, Co2+ and Mn2+ were stimulatory. Zn2+ and Cu2+ suppressed the activity. Concanavalin A, a plant lectin, was markedly inhibitory, suggesting that a glycoprotein moiety was necessary to express enzyme activity. Ecto-5'-nucleotidase activity was not modified during phagocytosis of serum-treated zymosan by mesangial cells. Rat cultured glomerular epithelial cells exhibited a 5'-nucleotidase activity which was 4 times lower than that of the mesangial cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines

Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5'-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30-40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5'-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II- and actually reduced by cN-I- overexpression. In H9c2 cells, in which 5'-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.     

Beta,gamma-methylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor

The mechanism underlying beta,gamma-methylene ATP (beta,gamma-MeATP)-induced cAMP elevation was investigated in rat glioma C6Bu-1 cells. Beta,gamma-MeATP increased forskolin-stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine-induced response, did not eliminate the beta,gamma-MeATP-induced response. However, combination of ADA with alpha,beta-methylene ADP (alpha,beta-MeADP), an ecto-5'-nucleotidase inhibitor, blocked the beta,gamma-MeATP-induced response. AMP, the substrate for ecto-5'-nucleotidase, also induced cAMP formation in a manner sensitive to XAC and alpha,beta-MeADP inhibition. However, the AMP-induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from beta,gamma-MeATP and AMP. In addition, alpha,beta-MeADP inhibited the conversion of beta,gamma-MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from beta,gamma-MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto-phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5'-nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT-PCR. These results suggest that C6Bu-1 cells possess ecto-enzymes converting beta,gamma-MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.