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1.
Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [14C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [14C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN
adenine nucleotide
- Chl
chlorophyll
- DTT
dithiothreitol
- FCCP
carbonylcianide p-trifluoromethoxyphenilhydrazone
- Pi
inorganic orthophosphate
- PMS
phenazine methosulfate
- TLC
thin-layer chromatography
- Tricine
N-[tris(hydroxymethyl)methyl] glycine 相似文献
2.
3.
Alteration of the nucleotide-binding site asymmetry of chloroplast coupling factor 1 by catalysis 总被引:1,自引:0,他引:1
Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule. 相似文献
4.
M Komatsu-Takaki 《Journal of biochemistry》1983,94(4):1095-1100
ADP binding brought about by inorganic phosphate addition (Pi-dependent ADP binding) on membrane-bound chloroplast coupling factor was studied and the following results were obtained. Under energization by illumination or by acid-base transition, Pi brought about the binding of ADP with an apparent Km value of 0.22 mM. This effect of Pi was lost rapidly after turning the light off or after acid to base transition, concomitant with the loss of ATP synthesizing activity. Pi-dependent ADP binding was inhibited by phlorizin to nearly the same extent as was ATP synthesis. The inhibitory effects of phlorizin on both the Pi-dependent ADP binding and ATP synthesis increased with the decrease of Pi concentration. These results suggest that the Pi-dependent ADP binding reaction participates in the ATP synthesis reaction and that phlorizin inhibits the P1 binding process. 相似文献
5.
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. 相似文献
6.
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. 相似文献
7.
G Girault G Berger J M Galmiche F Andre 《The Journal of biological chemistry》1988,263(29):14690-14695
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. 相似文献
8.
We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change. 相似文献
9.
Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner. 相似文献
10.
Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl2, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the
. In the absence of Mg2+, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg2+ present. In the presence of nigericin, Mg2+, and ATP, inactivation occurs after a 10–15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding. 相似文献
11.
Inhibitors of cytoplasmic polyribosome function and chloroplastpolyribosome function were used to study the site of synthesisof the five subunits of coupling factor I (CF1) in Pisum sativum.The results of these in vivo experiments are presented as evidencefor the cytoplasmic synthesis of two subunits, C and D, andthe synthesis in the chloroplast of the other three, A, B andE.
1 Supported in part by grant PCM-74-13534 from the NationalScience Foundation. (Received April 17, 1978; ) 相似文献
12.
Malyan AN 《Biochemistry. Biokhimii?a》2002,67(11):1253-1257
A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF1 was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF1 -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF1 with Mg2+ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k
d
(ATP) 10–3 min–1. Noncatalytic sites of CF1 were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF1 with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites. 相似文献
13.
14.
Periodate-oxidized ADP, if left in aqueous solution, loses its phosphates by beta-elimination. This dephosphorylated dialdehyde compound caused rapid and irreversible inhibition of membrane-bound spinach chloroplast coupling factor 1 (CF1). Inhibition was 2.5 times faster in the light than in the dark. A high concentration of uncoupler eliminated the light stimulation. Light could be replaced by an acid-base transition. Therefore, the dialdehyde reacts with a site or sites on CF1 that become exposed by a high-energy state-induced conformational change. The substrate nucleotides ADP, ATP, GDP, and GTP protected against inhibition while Pi and the non-substrate nucleotides AMP, GMP, CTP, and UTP did not. The protection by GTP was competitive and magnesium-dependent, suggesting that the dialdehyde binds to a nucleotide-binding site. However, the corresponding UDP and CDP dialdehyde derivatives also inhibited CF1 and showed the light-stimulation effect, indicating that the adenine is not important for the binding. These derivatives could be binding to a nucleotide-binding site or to another reactive site that becomes exposed during the light-induced conformational change. In the latter case the protection by substrate nucleotides would be due to prevention of the energy-dependent conformational change. 相似文献
15.
1. Divalent antibodies against chloroplast coupling factor 1 inhibited the factor ATPase, ATP synthesis, hydrolysis and Pi-ATP exchange in chloroplasts. These antibodies also inhibited coupled electron flow rates but not the basal or uncoupled rates. 2. Several types of non-precipitating, modified antibodies prepared from the original antibody preparation strongly inhibited the ATPase and Pi-ATP exchange reaction but had little effect on ATP formation. 3. It is suggested that the inhibition of ATP synthesis by the divalent antibodies is probably due to an indirect blocking of the active site, while the inhibition of ATP-utilizing reactions by the modified antibodies is related to their effect on the transfer of ATP from a non-catalytic to a catalytic site on coupling factor 1, via an energy-dependent conformational change. 相似文献
16.
Malyan AN 《Biochemistry. Biokhimii?a》2007,72(7):728-734
This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more. 相似文献
17.
Mizuho Komatsu-Takaki 《FEBS letters》1984,175(2):433-438
Decay of light-triggered ATP hydrolysis in the dark was diminished with a decrease in chloroplast concentration. The enhancing effect of NH4Cl on ATP hydrolysis decreased with dark time. The decrease was much faster than that in ATP hydrolysis activity. The NH4Cl effect increased with ATP preincubation time. Reactivation of ATP hydrolysis occurred with the progress of ATP hydrolysis. Pi enhanced the activation remarkably. These results suggest that ATP hydrolysis produces some energized state, which stimulates NH4C1 effect and makes coupling factor active in the presence of Pi and that to keep coupling factor active, energy is not necessarily needed. 相似文献
18.
Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1. 总被引:4,自引:0,他引:4
A B Shapiro K D Gibson H A Scheraga R E McCarty 《The Journal of biological chemistry》1991,266(26):17276-17285
Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have been mapped by fluorescence resonance energy transfer distance measurements (see McCarty, R. E., and Hammes, G. G. (1987) Trends Biochem. Sci. 12, 234-237). Using the same technique, we mapped the location of nucleotide-binding site 4, a tight, exchangeable site (Shapiro, A. B., Huber, A. H., and McCarty, R. E. (1991) J. Biol. Chem. 266, 4194-4200). Two arrangements of the energy transfer map of coupling factor 1 were found which are compatible with the available data. The two arrangements make different predictions about which sites interact in cooperative catalysis. 相似文献
19.
Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors. 相似文献
20.
Kawakami H Ozaki S Suzuki S Nakamura K Senriuchi T Su'etsugu M Fujimitsu K Katayama T 《Molecular microbiology》2006,62(5):1310-1324
Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp. 相似文献