Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.     

Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.     

Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1)N-4-Azido-2-nitrophenyl-gamma-[3H]aminobutyryl-AdoPP[NH] P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of [3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]P/mol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the alpha- and beta- subunits of F1. At low concentrations (less than 10 microM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the alpha-subunit; at concentrations equal to, or greater than 75 microM, both alpha- and beta-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (alpha or beta), suggesting that each of the two subunits, alpha and beta, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The precentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggestion that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.     

Localization of the high-affinity binding site for ATP on the membrane-bound chloroplast ATP synthase

The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.     

Binding and hydrolysis of 2-azido-ATP and 8-azido-ATP by isolated mitochondrial F1: characterisation of high-affinity binding sites

The kinetic parameters for the hydrolysis by F1 of the photoreactive nucleotide analogue 2-azido-ATP were determined (Vmax, 105 U/mg F1; Km, 250 microM, in the presence of 1.0 mM SO2-3). In the absence of an activating anion, a non-linear relationship in a Lineweaver-Burk plot was found for the hydrolysis of 2-azido-ATP. The 2-azido-analogues of ATP and ADP proved to be good photoaffinity labels causing notable inactivation of the F1-ATPase activity upon irradiation at 360 nm. This inhibition was also used to demonstrate high-affinity binding of these analogues to a catalytic binding site on the F1. High-affinity binding proved to be an Mg2+-requiring process, occurring with both 2-azido-ATP and 2-azido-ADP but hardly or not occurring with 8-azido-AT(D)P. Covalent binding of 2-nitreno-ATP upon irradiation of F1 containing tightly bound [beta-32P]2-azido-ATP results in a proportional inhibition of ATPase activity, extrapolating to 0.92 mol of covalently bound label per mol of F1 needed for the complete inactivation of the enzyme. When the F1 was irradiated in the presence of excess [beta-32P]2-azido-AT(D)P, 3-4 mol of label were bound when the enzyme was fully inactivated. In all cases, all or most of the radioactivity was found on the beta subunits.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites

A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAPDAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the alpha- and beta-subunits. The apparent Ki for the photoaffinity analogue was 1.40 +/- 0.17 microM (mean +/- SD, n = 3) as measured by competition with [3H] colchicine. Values of the apparent KdS for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 +/- 0.11 microM and 11.6 +/- 3.5 microM (mean +/- SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 +/- 0.06 and 1.3 +/- 0.4 mol/mol of tubulin (mean +/- SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25 degrees C, 15 +/- 3 mol % (mean +/- SD, n = 6) of the [3H]NAPDAC was covalently bound to the alpha-subunit, and 67 +/- 9 mol % (mean +/- SD, n = 6) was covalently bound to the beta-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both alpha- and beta-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled alpha- and beta-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the alpha-subunit and in five peptides of the beta-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.     

Tightly bound 2-azido-adenine nucleotides at catalytic and noncatalytic sites of the rat liver F1 ATPase label adjacent tryptic peptides of the beta subunit

UV irradiation of rat liver F1 ATPase, previously exposed to Mg2+ and [beta, gamma-32P]-2-azido-ATP and separated from medium nucleotides, covalently modifies two tyrosine residues in adjacent tryptic peptides of the beta subunit. This results from the occupancy by 2-azido-ATP or 2-azido-ADP of two distinct types of nucleotide binding sites, the catalytic and noncatalytic sites. The two modified peptides are identical to the ones modified by 2-azido-adenine nucleotides in the beef heart F1 ATPase. Both catalytic and noncatalytic sites are labeled when the ATPase is exposed to [beta-32P]-2-azido-ADP in the presence or the absence of 5'-adenylyimidodiphosphate (AMP-PNP), showing that two distinct types of ADP binding sites are present on the liver enzyme. Similar incorporation of 2-azido-adenine nucleotides is obtained when membrane-bound rat liver F1 ATPase is incubated with Mg2+ and [beta, gamma-32P]-2-azido-ATP.     

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.     

Photolabeling of the phosphate binding site of mitochondrial F1-ATPase by [32P]azidonitrophenyl phosphate. Identification of the photolabeled amino acid residues

[32P]Azidonitrophenyl phosphate [( 32P]ANPP) is a photoactivatable analogue of Pi. It competes efficiently with Pi for binding to the F1 sector of beef heart mitochondrial ATPase and photolabels the Pi binding site located in the beta subunit of F1 [Lauquin, G. J. M., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626]. By cleavage of the photolabeled beta subunit of F1 with cyanogen bromide, trypsin, and chymotrypsin, bound [32P]ANPP was localized in a fragment spanning Thr 299-Phe 326. By Edman degradation of the radiolabeled tryptic peptide spanning Ile 296-Arg 337, [32P]ANPP was found to be attached covalently by its photoreactive group to Ile 304, Gln 308, and Tyr 311. These results are discussed in terms of a model in which the phosphate group of [32P]ANPP interacts with a glycine-rich sequence of the beta subunit, spanning Gly 156-Lys 162, which is spatially close to the photolabeled Ile 304-Tyr 311 segment of the same subunit.     

Total number and differentiation of nucleotide binding sites on mitochondrial F1-ATPase. An approach by photolabeling and equilibrium binding studies

In this study 3'-O-[3-(4-azido-2-nitrophenyl)propionyl]-ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide-depleted F1-ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki = 50 microM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between alpha and beta subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3'-ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows. The total number of sites which can be simultaneously occupied by nucleotides on F1 is six. Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727] that the high-affinity sites are the catalytic ones which can be covalently labeled by 3'-O-[5-azidonaphthoyl(1)]-ADP [Lübben, M., Lücken, U., Weber, J. & Sch?fer, G. (1984) Eur. J. Biochem. 143, 483-490], it appears likely that azidonitrophenylpropionyl-ADP is a specific photolabel for the lower-affinity sites on nucleotide-depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs. The labeled low-affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.     

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF1): the effect on the CF1-ATPase activity

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19-24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[beta-32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the beta subunit of the CF1 and since there are probably three beta subunits per CF1, these results indicate strong cooperativity among beta subunits and between the site of tightly bound nucleotides and the catalytic sites.     

Functionally similar vanadate-induced 8-azidoadenosine 5'-[alpha-(32)P]Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.     

Localization of the tight ADP-binding site on the membrane-bound chloroplast coupling factor one

The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.     

Identification of amino acid residues photolabeled with 2-azido[alpha-32P]adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase

When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido[alpha-32P]adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled [Dalbon, P., Boulay, F., & Vignais, P. V. (1985) FEBS Lett. 180, 212-218]. The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.     

Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.     

Is pyrophosphate an analog of adenosine diphosphate for beef heart mitochondrial F1-ATPase

Beef heart mitochondrial F1 possesses three pyrophosphate-binding sites, which comprises one high affinity binding site (Kd approximately equal to 1 microM) and two lower affinity sites (Kd approximately equal to 20 microM). High affinity pyrophosphate binding required the presence of Mg2+ in the incubation medium. Pyrophosphate competed with ADP, but not with Pi for binding to mitochondrial F1. Upon binding of 3 mol of pyrophosphate/mol of F1, one of the three tightly bound nucleotides present in native F1 was released. Like ADP and in contrast to Pi, pyrophosphate enhanced the fluorescence intensity of F1-bound aurovertin, and it prevented the photolabeling of F1 by 2-azido-ADP. As aurovertin and 2-azido-ADP are ligands of the beta subunit of F1, it is likely that pyrophosphate binds preferentially to the beta subunit. Whereas the binding affinity of F1 for Pi was increased by concentrations of pyrophosphate lower than 100 microM, it was decreased by a higher concentration of pyrophosphate. This biphasic effect of pyrophosphate on Pi binding was not observed with ADP, which, at all concentrations tested, inhibited Pi binding. Except for the effect of pyrophosphate on Pi binding to F1, for all the other effects, pyrophosphate mimicked ADP. It is suggested that pyrophosphate and ADP share the same binding site on F1 and that pyrophosphate interacts with the same amino acid residues as those interacting with the alpha and beta phosphate groups of ADP.