Biomedical subjects

T Hisabori

Publications and source records attributed to T Hisabori.

At least 37 records · Page 2Linked to original sources

PubMed1998

The beta subunit of chloroplast ATP synthase (CF0CF1-ATPase) is phosphorylated by casein kinase II.

We studied a phosphate acceptor for casein kinase II (CK-II) in chloroplasts, and found a 56 kDa protein (p56) as an acceptor, which was partially purified from the stroma of spinach chloroplasts. The N-terminal amino acid sequence of p56 was identical with that of the beta subunit of chloroplast ATP synthase (CF0CF1-ATPase). In addition, the recombinant beta subunit of CF1 was phosphorylated when the subunit was incubated with CK-II. These results suggest that the beta subunit of CF1 is a substrate protein of CK-II in the chloroplast.

Amino Acid Sequence↗

PubMed1997

Thermophilic F1-ATPase is activated without dissociation of an endogenous inhibitor, epsilon subunit.

Subunit complexes (alpha3beta3gamma, alpha3beta3gammadelta, alpha3beta3gammaepsilon, and alpha3beta3gammadeltaepsilon) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis. The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of epsilon subunit was more profound. The (-epsilon) complexes, alpha3beta3gamma and alpha3beta3gammadelta, initiated ATP hydrolysis without a lag. In contrast, the (+epsilon) complexes, alpha3beta3gammaepsilon and alpha3beta3gammadeltaepsilon, started hydrolysis of ATP (<700 microM) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP. Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+epsilon) complexes bound substrate only slowly. Differing from Escherichia coli F1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit. This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Adenosine Triphosphate↗

PubMed1997

The regulatory functions of the gamma and epsilon subunits from chloroplast CF1 are transferred to the core complex, alpha3beta3, from thermophilic bacterial F1.

The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated. Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of epsilon(c) or epsilon(t) to the alpha3beta3gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.

Bacillus↗

PubMed1996

Catalytic activities of alpha3beta3gamma complexes of F1-ATPase with 1, 2, or 3 incompetent catalytic sites.

In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha3beta3gamma complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu.Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit. The Glu.Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha3beta3gamma complexes, one containing beta(wild-type) and the other Glu.Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha3beta3gamma complexes were reconstituted. Each of the complexes containing a different number of Glu.Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M.(1989) J. Biochem. (Tokyo) 106, 730-734).

Adenosine Diphosphate↗

PubMed1996

Structural asymmetry of F1-ATPase caused by the gamma subunit generates a high affinity nucleotide binding site.

The alpha 3 beta 3 gamma and alpha 3 beta 3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the alpha 3 beta 3 complex. The results of equilibrium dialysis show that the alpha 3 beta 3 gamma complex has a high affinity nucleotide binding site and several low affinity sites, whereas the alpha 3 beta 2 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the beta subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the alpha 3 beta 3 gamma complex is accelerated by the chase addition of excess ATP, whereas that by the alpha 3 beta 3 complex is not. We further examined the complexes containing mutant beta subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant beta subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K, Yutani, K., and Yoshida, M. (1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the beta subunit in the mutant alpha 3 beta 3 gamma complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant alpha 3 beta 3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the gamma subunit is responsible for the generation of a high affinity nucleotide binding site on the beta subunit in F1-ATPase where cooperative catalysis can proceed.

Adenosine Diphosphate↗

PubMed1996

The heterogeneous interaction of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli.

The interactions of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli (EF1) were examined and compared with those in the case of mitochondrial F1-ATPase (MF1) and F1-ATPase from thermophilic Bacillus PS3 (TF1). EF1 hydrolyzed substoichiometric TNP-ATP faster than TF1 or MF1, although some 20% of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF1 for the product, TNP-ADP, was weaker than that of TF1 or MF1, and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF1 was smaller than that of MF1 or TF1 under similar experimental conditions. When an excess amount of TNP-ATP was added to EF1 and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated beta subunit of TF1, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions described in this paper.

Adenosine Triphosphate↗

PubMed1994

Catalytic cooperativity of beef heart mitochondrial F1-ATPase revealed by using 2',3'-O-(2,4,6-trinitrophenyl)-ATP as a substrate; an indication of mutually activating catalytic sites.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bovine mitochondrial F1-ATPase (MF1) was examined under substoichiometric and stoichiometric conditions to investigate the relationship between the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis. The hydrolysis of bound TNP-ATP under substoichiometric condition proceeded slowly, with a first order rate constant of 0.014 s-1. However, hydrolysis was greatly accelerated by addition of excess ATP. The hydrolyzed product, TNP-ADP, did not dissociate from the enzyme even after the addition of excess ATP. These properties were the same for both native and nucleotide depleted enzyme. The difference spectrum induced by binding TNP-ATP to MF1 had a distinct peak at 410 nm and a deep trough at 395 nm, which were similar to those induced when TNP-ATP bound to the isolated beta subunit of the thermophilic F1-ATPase. The magnitude of difference spectra as a function of TNP-ATP concentration suggested the presence of at least two types of binding sites on the MF1 molecule. The first site, where substoichiometric TNP-ATP was hydrolyzed, had a very high affinity for TNP-ATP. TNP-AT(D)P bound to this site did not dissociate even in the presence of excess ATP. TNP-AT(D)P bound to the second site dissociated slowly when excess ATP was added. The steady state ATPase activity at 100 microM ATP was linearly suppressed as pre-loaded TNP-ATP increased. The binding of 2 mol of TNP-ATP per mol of MF1 was required to abolish ATPase activity. A model which assumes mutually-activating two catalytic sites is presented to explain these results.

Adenosine Triphosphate↗

PubMed1994

Asymmetry of the three catalytic sites on beta subunits of TF1 from a thermophilic Bacillus strain PS3.

F1-ATPase isolated from plasma membrane of a thermophilic Bacillus strain PS3 (TF1) has very little or no endogenously bound adenine nucleotides. However, it can bind one ADP per mol of the enzyme on one of three beta subunits to form a stable TF1.ADP complex when incubated with a high concentration of ADP [Yoshida, M. & Allison, W.S. (1986) J. Biol. Chem. 261, 5714-5721]. The same TF1.ADP complex was recovered after filling all ADP binding sites with [3H]ADP and repeated gel filtration. Direct binding assay revealed that the TF1.ADP complex had lost the highest affinity site for TNP-ADP. When a substoichiometric amount of TNP-ATP was added, the complex hydrolyzed TNP-ATP slowly (single site hydrolysis), like native TF1. However, this hydrolysis was not promoted by chase-addition of excess ATP. The optimal pH of the ATPase activity of TF1 or the TF1.ADP complex measured with a short reaction period, 6.5, was lower than the reported value, 9.0, under the steady-state condition. Although the bound ADP was released from the complex only when the enzyme underwent multiple catalytic turnover, the rate of this release was much slower than the turnover. These results suggest that when one ADP binds to a site on one of the beta subunits and stays there for a long time, the enzyme will change form and the bound ADP will become a special species which is not able to be directly involved in the enzyme catalysis. This binding site for ADP appears to be the first site responsible for the single-site catalysis reaction observed for native TF1.

Adenosine Diphosphate↗

PubMed1993

AT(D)PMg-induced dissociation of the alpha 3 beta 3 complex of the F1-ATPase from a thermophilic Bacillus PS3 into alpha 1 beta 1 heterodimers is prevented by mutation beta (Y341C).

AT(D)PMg induces dissociation of the alpha 3 beta 3 complex of F1-ATPase from a thermophilic Bacillus strain. PS3, into the alpha 1 beta 1 heterodimers [(1991) Biochim. Biophys. Acta 1056, 279-284] but the location of the AT(D)PMg binding site responsible is not known. From the analysis of AT(D)PMg binding properties of the isolated mutant beta subunit, beta(Y341C), and the stability of the alpha 3 beta(Y341C)3 complex in the presence of AT(D)PMg, we conclude that binding of AT(D)PMg to the Tyr-341 site of the beta subunit(s) in the alpha 3 beta 3 complex triggers the dissociation of the alpha 3 beta 3 complex into the alpha 1 beta 1 heterodimers.

Adenosine Diphosphate↗

PubMed1993

Effect of covalent binding of a derivative of 2',3'-O-(2,4,6-trinitrophenyl)-ADP to the tight binding site of CF1 on the enzyme activity.

Irradiation of isolated chloroplast thylakoids with TNP-ADP results in non-covalent binding and covalent incorporation of a reaction product of TNP-ADP formed by photosynthetic reduction into the so-called "tight" nucleotide binding site of CF1 [Ponse et al. (1992) Z. Naturforsch. 47c, 264-274]. CF1 extracted from thus-loaded thylakoid membranes yielded maximal incorporation of 1 mol/mol of CF1. Almost half had the covalent bond with CF1. In experiments with TNP-[14C]ADP, radioactivity was detected almost equivalently on alpha and beta subunits, suggesting that the binding site may be at the interface between alpha and beta subunits. Enzyme activities of the thylakoid membrane-bound enzyme after covalent labeling were measured. Inhibition, ranging from 20 to 25%, was less than expected from the percentage of CF1 molecules labeled (40-50%). It is suggested that only half of the labeled enzymes, probably those with the nucleotide analog linked to the beta subunit, are inactive.

Adenosine Diphosphate↗

PubMed1993

Magnesium regulates both the nucleotide binding and the enzyme activity of isolated chloroplast coupling factor 1.

The inactivation and the activation of the ATPase of isolated CF1 as assayed by the hydrolysis of ATP in 10 s depended on prior binding of ADP-Mg and ATP-Mg. The effects of Mg2+ on the nucleotide binding kinetics were studied by monitoring the time courses of UV spectral changes induced by the interaction between CF1 and ADP or ATP using a rapid-scan spectrophotometer equipped with a stopped-flow cell. The apparent rate constant of ADP binding to the two high-affinity sites on CF1 (designated sites B and C in the previous report [Hisabori, T. & Sakurai, H. (1984) Plant Cell Physiol. 25, 483-493]) was drastically increased by prior binding of Mg2+ to CF1, but not ATP. The inhibitory effect of Mg2+ was attributed to a marked increase in kon for the inhibitory ADP binding at the high-affinity sites induced by the previous binding of Mg2+ to the enzyme. The location of site B is suggested to be on the beta subunit based on the difference spectral change induced by binding of the ADP analog 2',3'-O-(2,4,6-trinitrophenyl)ADP to CF1.

Adenosine Diphosphate↗

PubMed1992

Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.

Adenosine Diphosphate↗

PubMed1991

Two-dimensional gel electrophoresis of the membrane-bound protein complexes, including photosystem I, of thylakoid membranes in the presence of sodium oligooxyethylene alkyl ether sulfate/dimethyl dodecylamine oxide and sodium dodecyl sulfate.

Two-dimensional polyacrylamide gel electrophoresis (PAGE), using a mixture of sodium oligooxyethylene alkyl ether sulfate and dimethyl dodecylamine oxide as detergents (AES-DDAO mixture) in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension, was developed and applied to an analysis of the photosystem I (PS I) complex in thylakoid membranes prepared from spinach chloroplasts. When thylakoid membranes of chloroplasts were solubilized directly in the AES-DDAO mixture and subjected to PAGE in the presence of these detergents as the first dimension, some protein complexes containing chlorophyll were observed. The protein components in these complexes separated into an array of polypeptide spots when the strip of gel after PAGE in the first dimension was subjected to PAGE in the presence of SDS as the second dimension. The main band of protein which separated in the first dimension was demonstrated to be the PS I complex. This complex retained the intrinsic photochemical activity of P700 even after it was subjected to one-dimensional PAGE. These results suggest that certain protein complexes can be separated, with the maintenance of their original structures, by electrophoresis in the presence of the AES-DDAO mixture, and this method appears to have valuable potential for analysis of the components of membrane-bound protein complexes.

Cell Membrane↗

PubMed1990

The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1.

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.

Amino Acid Sequence↗

PubMed1989

The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase.

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.

Adenosine Diphosphate↗

PubMed1989

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active.

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.

Adenosine Diphosphate↗

PubMed1988

Site-directed mutagenesis of stable adenosine triphosphate synthase.

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.

Adenosine Diphosphate↗

PubMed1986

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Adenosine Diphosphate↗