The nucleotide binding site of F1-ATPase which carries out uni-site catalysis is one of the alternating active sites of the enzyme

Nucleotide-depleted mitochondrial F1-ATPase binds 3'-(2')-O-(2-nitro-4-azidobenzoyl)-derivatives of ATP (NAB-ATP) and GTP (NAB-GTP) when these nucleotide analogues are added to the enzyme in equimolar quantities in the presence of Mg2+ (uni-site catalysis conditions). The binding of NAB-ATP is accompanied by its hydrolysis and inorganic phosphate dissociation from the enzyme; NAB-ADP remains bound to F1-ATPase. The F1-ATPase X NAB-ADP complex has no ATPase activity and its reactivation in the presence of an excess of ATP is accompanied by NAB-ADP release. The illumination of the F1-ATPase complexes with NAB-ADP or NAB-GDP leads to the covalent binding of one nucleotide analogue molecule to the enzyme and to the irreversible inactivation of F1-ATPase. It follows from the results obtained that the modification of just one of the F1-ATPase catalytic sites is sufficient to complete the inhibition of ATPase activity.     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites.

Inactivation of the isolated ATPase portion of ATP synthase from beef-heart mitochondria (F1) by its natural inhibitor protein (IP) during steady-state ATP hydrolysis is accompanied by a trapping of 1 mol nucleotide/mol F1 in one of the catalytic sites. The trapped nucleotide is not released during incubation of IP-inhibited F1 in the presence of MgATP at pH 8.0 for at least 20 min, indicating a very low turnover rate of the IP.F1 complex. The ATP/ADP ratio of the trapped nucleotides is higher than that found for transitorily bound nucleotides under the same conditions but in the absence of IP. The IP impairs the acceleration of ATP hydrolysis and product release steps that results from the binding of ATP to an alternate catalytic site. It also inhibits ATP hydrolysis by a single catalytic site or shifts the equilibrium toward ATP formation from bound ADP and Pi. At high pH, an active acidic form of the free IP is transformed to the inactive basic one with a half-time of 3-4 s. This process seems to be prevented by IP binding to F1. The inactive basic form of IP does not compete with the active acidic IP for the binding to F1. The data do not favor the existence of a long-lived catalytically active IP.F1 intermediate during IP action on F1. The reactivation of IP-inhibited membrane-bound F1 by energization may be due to a conformational change in the IP.F1 complex allowing the transformation of IP into an inactive basic state that rapidly dissociates.     

Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds

The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP.     

Communications between the high-affinity cyclic nucleotide binding sites in E. coli cyclic AMP receptor protein: effect of single site mutations

The binding of adenosine 3',5'-cyclic monophosphate (cAMP) and its nonfunctional analogue, guanosine 3',5'-cyclic monophosphate (cGMP), to the adenosine 3',5'-cyclic monophosphate receptor protein (CRP) from Escherichia coli was investigated by means of fluorescence and isothermal titration calorimetry (ITC) at pH 7.8 and 25 degrees C. A biphasic fluorescence titration curve was observed, confirming the previous observation reported by this laboratory (Heyduk and Lee (1989) Biochemistry 28, 6914-6924). However, the triphasic titration curve obtained from the ITC study suggests that the cAMP binding to CRP is more complicated than the previous conclusion that CRP binds sequentially two molecules of cAMP with negative cooperativity. The binding data can best be represented by a model for two identical interactive high-affinity sites and one low-affinity binding site. Unlike cAMP, the binding of cGMP to CRP exhibits no cooperativity between the high-affinity sites. The effects of mutations on the bindings of cAMP and cGMP to CRP were also investigated. The eight CRP mutants studied were K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L. These sites are located neither in the ligand binding site nor at the subunit interface. The binding was monitored by fluorescence. Although these mutations are at a variety of locations in CRP, the basic mechanism of CRP binding to cyclic nucleotides has not been affected. Two cyclic nucleotide molecules bind to the high-affinity sites of CRP. The cooperativity of cAMP binding is affected by mutation. It ranges from negative to positive cooperativity. The binding of cGMP shows none. With the exception of the T127L mutant, the free energy change for DNA-CRP complex formation increases linearly with increasing free energy change associated with the cooperativity of cAMP binding. This linear relationship implies that the protein molecule modulates the signal in the binding of cAMP, even though the mutation is not directly involved in cAMP or DNA binding. In addition, the significant differences in the amplitude of fluorescent signal indicate that the mutations also affect the surface characteristics of CRP. These results imply that these mutations are not perturbing specific pathways of signal transmission. Instead, these results are more consistent with the concept that CRP exists as an ensemble of native states, the distribution of which can be altered by these mutations.     

pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains

Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.     

Detecting precatalytic conformational changes in F1-ATPase with 4-benzoyl(benzoyl)-1-amidofluorescein, a novel fluorescent nucleotide site-specific photoaffinity label

A novel photoaffinity label for studies with the F1-ATPase has been synthesized and found to be an effective reporter of subunit conformational changes that occur in this enzyme upon multiple nucleotide-binding site occupancy. The new probe, 4-benzoyl(benzoyl)-1-amidofluorescein (BzAF), which possesses structural similarity to purine nucleotides, exhibits bifunctional characteristics that enable it to bind covalently to the exchangeable nucleotide sites on beef heart F1 (via photoactivation of the benzophenone moiety) and, once covalently linked, emit environmentally sensitive fluorescence (via selective excitation of the fluorescein moiety). BzAF binds competitively with ATP in the absence of illumination, with a KI of 50 microM. Under actinic irradiation necessary for generating the covalently reacting diradical triplet state of benzophenone, BzAF behaves as a nucleotide site-directed photoaffinity label of exchangeable (catalytic) sites, and the resulting photoinhibition of ATPase activity displays pseudo first-order rate-saturation kinetics that support formation of a dissociable BzAF.F1 complex (k-1/k1 = 58 microM) prior to covalent binding. The BzAF-induced photoinactivation is protectable with native nucleotide ligand (e.g. MgADP, Kprotect = 0.4 mM). Added corroboration of a catalytic cooperativity mechanism for F1 was obtained by finding a molar stoichiometric ratio [( 3H]BzAF:F1) of 1 required for complete inhibition of ATPase activity. Steady-state fluorescence studies with a unisite-labeled BzAF.F1 complex (a catalytically inactive species on which at least one exchangeable nucleotide-binding site remains unoccupied) display a saturable fluorescence quenching of the bound fluorescein upon titration with MgADP, but no change with MgAMP. These data imply that the filling of more than one of the catalytic binding sites/mol of F1 with nucleotide signals a precatalytic conformational adjustment that is transmitted between catalytic sites and across the beta-alpha-beta domain of the enzyme's subunit structure.     

Characterization of nucleotide binding sites on chloroplast coupling factor 1.

A study of the equilibrium binding of ADP, 1,N6-ethenoadenosine diphosphate, adenylyl imidodiphosphate, and 1,N6-ethenoadenylyl imidodiphosphate to solubilized spinach chloroplast coupling factor 1 (CF1) has been carried out. All four nucleotides were found to bind to two apparently identical "tight" sites, with characteristic dissociation contants generally less than 10 muM. The binding to these "tight" sites is similar in the presence of Mg2+ and Ca2+, is stronger in 0.1 M NaC1 than in 20 mM Tris-C1, and is only slightly altered by heat activation. The slow rate of association of ADP and 1,N6-ethenoadenosine diphosphate at these sites rules out the possibility that they are catalytic sites for ATPase activity on the solubilized enzyme. A third tight site for adenylyl imidodiphosphate was found on the heat-activated enzyme. The dissociation constant for this interaction (7.6 muM) is similar to the adenylyl imidodiphosphate competitive inhibition constant for ATPase activity (4 muM). ADP, which inhibits ATPase activity but is not a strong competitive inhibitor, binds only weakly at a third site (dissociation constant greater than 70 muM). One mole of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted per mole of CF1 prevents ADP and adenylyl imidodiphosphate binding at the "catalytic" site and abolishes the ATPase activity. A model is proposed in which the "tight" nucleotide binding sites act as allosteric conformational switches for the ATPase activity of solubilizedCF1.     

Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.     

Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G., Roux, B., & Gautheron, D.C. (1991) Biochemistry 30, 3256-3262]. The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites. Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength. On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation. High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one. In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site. The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding. In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent. The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide. ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide.     

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.     

A fluorescence investigation of the nucleotide binding sites of the Ca ATPase

Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.     

Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits.     

Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa.

1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.     

Effect of nucleotides and inhibitory anions on mitochondrial ATPase. Its pH dependence

The effect of pH on the sensitivity of F1-ATPase as well as mitochondrial ATPase activity to nucleoside diand triphosphates and to inhibitory anions such as cyanate and thiocyanate, has been studied. The results obtained show that nucleotides could act as activators or inhibitors of the ATPase hydrolytic activity depending on pH, substrate concentration, and binding of the enzyme to the membrane. The effect of those nucleotides which activate the hydrolysis of ATP-Mg2+ was more pronounced beyon the optimum pH corresponding to each of the three catalytic sites of the enzyme, whereas those which are inhibitors had a lower effect above this value. The sensitivity to the inhibitory anions decreased with increasing pH values; the decrease in the inhibitory effect was sharper when approaching the optimum pH value. These data are in agreement with the existence in mitochondrial ATPase of two different regulatory sites, one being specific for binding nucleotides, and another for anions. Both of them showed a different response upon changes of pH.     

Kinetic analysis of hydrolysis of free ATP and MgATP by natural actomyosin]

Computer analysis of experimental data published in 1-3 allowed to establish the presence of two non-interacting inequivalent hydrolytic sites in actomyosin molecule, one of them being specific for binding and hydrolysis of free ATP, the other--for MgATP. Thus both species of ATP are the substrates of actomyosin ATPase. Actomyosin molecule seems to bind on more (in additon to two active sites) substrate molecule (MgATP) at some non-catalytic regulatory site. The formation of the enzyme-substrate complex having three ATP molecules (one molecule of free ATP and two--of MgATP) is accompanied by the loss of the activity. An approach to the research of kinetic equations for complex systems considerably decreasing a number of variations to consider is given in this work.     

The binding of aurovertin to isolated F1 (mitochondrial ATPase).

1. Isolated F1 contains 14.9% N, indicating the presence of at least 8% non-protein material. The Lowry method, standardized with bovine serum albumin, correctly measures the protein content. 2. An extinction coefficient of 28.5 mM-1.cm-1 at 367.5 nm was found for aurovertin D in ethanol. 3. The fluorescence enhancement of aurovertin bound to F1 at pH 7.5 was found to be more than 100-fold. 4. Binding parameters calculated from the fluorescence enhancement with fixed F1 and variable aurovertin concentrations, and vice versa, indicate two binding sites per F1 molecule. 5. The fluorescence data are not readily interpreted on the basis of successive binding of aurovertin by 3-component binding reactions of the form E + A in equilibrium EA, but fit closely a model of two non-interacting sites binding aurovertin in a 4-component reaction, EF + A in equilibrium EA + F, with an equilibrium constant of about 2.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase.

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.