High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei.

First molecular characterisation of a Brazilian Burkholderia mallei strain isolated from a mule in 2016.

We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.

First Draft Genome for a Burkholderia mallei Isolate Originating from a Glanderous Mule from Brazil.

Burkholderia mallei is the etiological agent of glanders. Here, we present the draft genome sequence of Burkholderia mallei strain 16-2438_BM#8 that was isolated from a mule found dead in Pernambuco, northeast Brazil. It is the first available genomic sequence from a strain isolated on the American continent.

Macromolecule-ligand binding studied by the Hummel and Dreyer method: current state of the methodology.

The use of the Hummel and Dreyer method to measure binding parameters of ligand-macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.

Use of the hummel and dreyer method for the study of nucleotide binding on chloroplast ATPase CF1.

The study of the binding of the nucleotides ADP and ATP on the exchangeable sites of chloroplast ATPase CF1 has been carried out by the Hummel and Dreyer method applied to HPLC. It has been shown that this method was well fitted to the problem: rapidity of exchange, absence of noticeable modification after binding, presence of a constant concentration of ligand during the chromatography, which stabilizes these low affinity complexes. The dissociation constants of binding of ADP, ATP and of their magnesium salt complexes have been determined. In order to measure the simultaneous binding of ADP and ATP when present in mixture, we have modified the method by using an anion-exchange column in place of the gel filtration column: the two nucleotides were easily separated, while the binding on the protein was unchanged. The extension of this method to the reversed-phase chromatography could also be considered for the binding of hydrophobic ligands.

Comparison of different cations (Mn2+, Mg2+, Ca2+) on the hydrolytic activity of chloroplast ATPase.

The influences of Mn2+, Mg2+, and Ca2+ on the enzymic activity of chloroplast ATPase have been compared, using an HPLC method for the separation of ADP. The dissociation constants of the divalent ion-ATP complexes have been determined by a spectrophotometric method, with the dye antipyrylazo III, and enzymic constants (dissociation constant of the ion-enzyme complexes, Michaelis constants, maximum rates) have been calculated. The comparison between the rates obtained, respectively, with Mn2+ and Ca2+ alone with that given by the mixture of these two ions, allows us to conclude that, as for Mg2+, Mn2+ is also an activator of chloroplast ATPase and that metal-free ATP is the true substrate.

Stability-related studies on 17D yellow fever vaccine.

Yellow fever (YF) vaccine using the 17D strain of YF attenuated virus has been produced at the Institut Pasteur in Dakar since 1962. Until now, the stabilised YF had an expiry date of utilization of two years from the end of the lot control process under storage at +4 degrees C. We conducted a stability study to assess the three full year validity of this preparation, when correctly stored at +4 degrees C to optimise the conditions of production, storage and availability of such a vaccine. The activity of 19 consecutive batches of vaccines kept for three years at +4 degrees C was compared to that of the same batches that were kept three years at -20 degrees C. Using the in vitro microculture method, we found that three-year storage at +4 degrees C induced a higher loss of activity than storage at -20 degrees C or than the accelerated degradation test of vaccines kept for 14 days at 37 degrees C. Whatever the conditions of storage, in all cases decreases in activity were below the WHO's requirements, i.e., < 1 log PFU/dose, and residual activity of the selected batches was over 1000 mouse LD50 per dose. We demonstrated that the 17D YF vaccine produced in Dakar has a shelf-life of three years and that its required potency was maintained at +4 degrees C, after reconstitution with saline diluent, following three-year storage at +4 degrees C.

[Reemergence of yellow fever in West Africa: lessons from the past, advocacy for a control program]. / Réémergence de la fièvre jaune en Afrique de l'Ouest: leçons du passé, plaidoyer pour un programme de contrôle.

In French speaking West Africa, yellow fever vaccine became compulsory in 1941 for the entire African and European population. From 1941 to 1960, 146 million doses were distributed and the number of yellow fever cases declined sharply. No case was reported from 1954 to 1960. As a result of an interruption in systematic immunization after 1960, ten major epidemics broke out in West Africa between 1965 and 1995 (over 200,000 cases and 40,000 deaths). In 1967, the WHO programme for eradication of smallpox was initiated and it mobilized WHO's energy and finances. The expanded programme of immunization (EPI) was initiated in 1977 but it did not include the yellow fever vaccine. In 1978, Primary Health Care advocated an immunization strategy through fixed health facilities. In 1986, to amend this strategy, WHO recommended accelerating EPI progress and instituting National Immunization Days (NIDs). In 1990, a recommendation was made to include the yellow fever vaccine in the EPI. In 1997, the target of global poliomyelitis eradication by the year 2000 reinforced the NID programme and led to the use of mobile teams. At a time when a measles eradication programme is going to take over from the poliomyelitis programme, we must firmly advocate not omitting the yellow fever vaccine as was the case in 1977. Indeed, in yellow fever endemic areas, WHO recommends a simultaneous association of yellow fever and measles vaccines for nine month-old infants. This opportunity must be seized to initiate a yellow fever control programme.

Cooperativity between the enzymatic sites of F1-ATPase revisited by the use of HPLC methods.

The fundamental question of the cooperativity between the enzymatic sites of F1-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF1, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF1 by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF1, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.

Binding sites for Mg(II) in H(+)-ATPase from Bacillus PS3 and in the alpha 3 beta 3 gamma subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes: a possible role for beta-His-324.

The binding sites for Mg2+ in wild type F1 ATPase (TF1) and in the alpha 3 beta 3 gamma subcomplex from the thermophilic bacterium Bacillus PS3 have been studied by EPR and by ESEEM and HYSCORE spectroscopy of complexes with the oxovanadium cation VO2+. Complexes of metal-depleted TF1 and substoichiometric amounts of VO2+ display low-temperature EPR signals with spectral parameters g parallel = 1.947 and g perpendicular = 1.980, and hyperfine couplings with 51V, A parallel = 169 x 10(-4) cm-1 and A perpendicular = 61 x 10(-4) cm-1, that are indicative of a binding site for VO2+ with nitrogen ligands from the protein. This binding site is probably identical with the metal binding site with strong affinity M1 that has been characterized using Mn2+ in a previous study [Buy, C., Girault, G., & Zimmermann, J. L. (1996) Biochemistry 35, 9880-9891]. The three-pulse ESEEM spectrum of the VO2+ complex with TF1 shows a frequency pattern with spectral properties that are evidence for two nitrogen ligands to the VO2+ with hyperfine couplings A1 = 4.75 MHz and A2 = 6.5 MHz and nuclear quadrupole parameters e2Qq1 = 2.8-3.2 MHz and e2Qq2 = 2.0-2.3 MHz. The ligands are identified as a lysine terminal amine and a histidine imidazole, which are proposed as Lys-164 and His-324 from a beta subunit. The HYSCORE data obtained for the VO.TF1 complex show correlations within each pair of the ESEEM nu dq peaks from the 14N nuclei, confirming the interpretation of the one-dimensional spectra. Evidence for the formation of a ternary complex by addition of VO2+ and ATP to metal-depleted TF1 is shown in the EPR and ESEEM spectra and in the contour plots of the HYSCORE data. Two pairs of correlation patterns are resolved in addition to the peaks from the two 14N ligands, which are interpreted as hyperfine couplings with 31P beta and 31P gamma of the ATP that binds the VO2+ cation. The assignment of the two hyperfine couplings to the specific phosphates, A(31P beta) = 15.5 MHz and A(31P gamma) = 8.7 MHz, in the VO.TF1.ATP complex is proposed by comparison with those measured for VO2+ in solution with ATP at pH 6.3 and 2.3. These results are discussed in light of the previous data with the analogous Mn.TF1 complex, and a model is proposed in which the native Mg2+ in the M1 site is coordinated by the side chain of beta-Lys-164 and is in close proximity to a histidine residue (probably beta-His-324) that may have a critical role. Additional coordination by two phosphates from ATP (probably the beta- and gamma-phosphates) is observed in the ternary complex VO.TF1.ATP. ESEEM and HYSCORE data are also obtained for the analogous complexes VO. alpha 3 beta 3 gamma and VO. alpha 3 beta 3 gamma .ATP that show very similar properties in terms of coordination of the divalent metal cation, except for the lysine ligand that is found to be lost in the ternary complex with ATP. It is suggested that this observation may reflect changes in the metal and nucleotide active sites that are associated with the absence of the delta and epsilon subunits in the subcomplex.

Synthesis, structure, and properties of MeSer1-tentoxin, a new cyclic tetrapeptide which interacts specifically with chloroplast F1 H(+)-ATPase differentiation of inhibitory and stimulating effects.

A new tentoxin analogue, in which the L-methyl alanine residue is substituted by L-methylserine, has been prepared following the synthetic pathway recently described for the synthesis of tentoxin [Cavelier, F., & Verducci, J. (1995) Tetrahedron Lett. 36, 4425-4428]. Using two-dimensional homonuclear proton nuclear magnetic resonance and structural analysis, we observed that MeSer1-tentoxin, like tentoxin, adopts several conformations in aqueous solution and presents self-aggregative properties. This analogue was found to be conformationally similar to the natural toxin. It showed the same efficiency as tentoxin in inhibition of ATPase activity of the isolated chloroplast F1 proton ATPase (CF1) as well as in inhibition of the ATP synthase activity of the membrane-bound enzyme (CF0CF1) in thylakoids and proteoliposomes. At concentrations above 10 microM, MeSer1-tentoxin did not reactivate CF1 to a high extent, contrary to tentoxin. It appeared, however, to bind in the same way, since the reactivating effect of tentoxin was inhibited by MeSer1-tentoxin. These results show that it is possible, using tentoxin analogues, to separate inhibitory and activating effects on the chloroplast ATPase, despite the limited chemical difference between the two toxins.

Tentoxin has at least two binding sites on CF1 and epsilon-depleted CF1 ATPases isolated from spinach chloroplast.

A new procedure for synthesis of 14C-labeled tentoxin [14C-MePhe[(Z)delta]3-tentoxin], with a high specific activity, is described. Binding experiments with CF1 or CF1-epsilon isolated from spinach chloroplast have been carried out using equilibrium dialysis technique. The results show the presence of two classes of binding sites. The association constants of the two major binding sites were derived from non-linear fitting of the binding curves. At 4 degrees C, the first binding site has a value of Ka1 = 8.2 x 10(5) M(-1) in CF1 and 8.7 x 10(5) M(-1) in CF1-epsilon, while the second binding site has lower affinity with Ka2 = 1.5 x 10(4) M(-1) in CF1 and 2.3 x 10(3) M(-1) in CF1-epsilon.

Metal binding sites of H(+)-ATPase from chloroplast and Bacillus PS3 studied by EPR and pulsed EPR spectroscopy of bound manganese(II).

The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe. After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP. TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment. Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported. The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1. The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment. The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios. For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule. This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation. For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P. From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors. It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal [Houseman, A. L. P., Morgan, L., LoBrutto, R., & Frasch, W. D. (1994) Biochemistry 33, 4910-4917]. Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand. This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex. Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz). An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5.1 MHz is observed after incubation of the ternary complex at room temperature. This is interpreted as a significant rearrangement of the coordination sphere of the Mn(II) in the M1 site of the Mn.TF1.ATP complex and may reflect conformational changes of catalytic significance that occur in the nucleotide binding site during unisite hydrolysis of ATP to ADP by this complex.

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin.

Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40 degrees C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitutions were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TFO-F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP x min(-1) x mg TF0F1(-1) in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) delta]3-Gly4)], as seen by two-dimensional 1H-nmr spectroscopy.

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) delta]3-Gly4)] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at -5 degrees C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180 degree rotation of a nonmethylated peptide bond.

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: study by high-performance liquid chromatography.

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima, Km values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of Km are widely different. The value obtained from the classical mechanism does not agree with KD, the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.

The prokaryotic thermophilic TF1-ATPase is functionally compatible with the eukaryotic CFo-part of the chloroplast ATP-synthase.

The ATP synthase from chloroplasts, CFo.F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP-dependent proton uptake was restored with these CFo-liposomes even better by the addition of the bacterial TF1-than of the related CF1-part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1-ATPase, in these CFo.F1-liposomes, but not in the hybrid CFo.TF1-liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo.TF1-liposomes and reconstituted CFo.F1-liposomes. These results indicate that the bacterial TF1-part binds to the eukaryotic CFo-part of four subunits forming a functional CFo.TF1-ATPase.

Photolabeling of the phosphate binding site of chloroplast coupling factor 1 with [32P]azidonitrophenyl phosphate.

Chloroplast F1-ATPase (CF1) was photolabeled by a radiolabeled photoactivatable derivative of Pi, 4-azido-2-nitrophenyl [32P]phosphate (ANPP). The radioactivity was localized in the beta subunit of CF1. Upon cleavage of the beta subunit by cyanogen bromide, the predominantly labeled peptide was recovered, which was subsequently subjected to tryptic digestion. A tryptic peptide (spanning Ile312-Arg354), was found to contain nearly all the covalently bound radioactivity. By Edman degradation, the labeled amino acid residues were identified as Tyr328, Val329 and Pro330. The labeled beta-Tyr328 of CF1 is the equivalent of beta-Tyr311 of F1 from beef heart mitochondria, which was previously found to be photolabeled by ANPP [J. Garin et al. (1989) Biochemistry 28, 1442-1448].

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts.

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.

Characterization of six nucleotide-binding sites on chloroplast coupling factor 1 and one site on its purified beta subunit.

By using gel filtration chromatography, following the technique of Hummel and Dreyer (Hummel, J., and Dreyer, W. (1962) Biochim. Biophys. Acta 63, 532-534), the adenine nucleotide-binding sites of isolated soluble chloroplast ATPase (CF1) and of the beta subunit were studied. CF1 possesses six adenine nucleotide-binding sites: two high affinity sites for ADP or ATP (KdH = 1-5 microM) in addition to one site where endogenous not-exchangeable ADP is bound, and three low affinity sites binding ADP or ATP with a dissociation constant (KdL = 15-20 microM) which is considerably increased in the presence of pyrophosphate. KdH is not modified by addition of pyrophosphate. The stability of nucleotide binding at the low affinity sites increases after heat activation of CF1. Removal of the delta or epsilon subunits on CF1 affects neither the number nor the binding parameters of the nucleotide-binding sites. The purified beta subunit possesses one easily exchangeable site/subunit. It is proposed that the low affinity sites on CF1 are the catalytic sites.