共查询到20条相似文献,搜索用时 0 毫秒
1.
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
2.
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
3.
The effect of pyrophosphate (PPi) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid
membranes, PPi competed with nucleotides for binding to noncatalytic sites. In the dark, PPi was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their
subsequent interaction with ADP and ATP. The effect of PPi on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PPi inhibited ATPase activity of ATP synthase. Addition of PPi to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated
its activity during prolonged incubation. These results indicate that the stimulating effect of PPi light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites.
The inhibition of ATP synthase results from competition between PPi and ATP for binding to catalytic sites.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 956–962. 相似文献
4.
Malyan AN 《Photosynthesis research》2006,88(1):9-18
A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k
d of about 5 min−1. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF0F1. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF1 dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF1 becomes bound to a thylakoid membrane. 相似文献
5.
Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF(1)) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF(1) with Mg(2+) produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k(-2)(ADP)=1.5 x 10(-1) min(-1) and k(-2)(ATP) congruent with 10(-3) min(-1), respectively. As follows from the study, the noncatalytic sites of CF(1) are not homogeneous. One of them retains the major part of endogenous ADP after CF(1) precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides. 相似文献
6.
Catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N6-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N6-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N6-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N6-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N6-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed. 相似文献
7.
The regulatory effects of malate on chloroplast Mg2+-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF1 Mg2+-ATPase activity. The subunit of CF1 may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg2+-ATPase was also controlled by the Mg2+/ATP ratio in the reaction medium. The enhancing effect of malate on Mg2+-ATPase activity depended on the presence of high concentrations of Mg2+ in the reaction mixture. Kinetic study showed that malate raised the Vmax of catalysis without affecting the Km for Mg2+ ATP. The experiments imply that the stimulation of Mg2+-ATPase by malate is probably correlated with the Pi binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF1
chloroplast coupling factor 1
- CF1 (-) and CF1 (-)
CF1 deficient in the and subunit
- MF1
mitochondria coupling factor 1
- NEM
N-ethylmaleimide
- PMS
phenazine methosulfate
- OG
n-octyl--d-glucopyranoside 相似文献
8.
In order to get insight into the origin of apparent negative cooperativity observed for F1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F1-ATPase, α(W463F)3β(Y341W)3γ and α(K175A/T176A/W463F)3β(Y341W)3γ. For α(W463F)3β(Y341W)3γ, apparent Km's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent Km's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α(K175A/T176A/W463F)3β(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent Km of ATPase activity (10 μM) roughly agreed with the highest Km of fluorescence measurements (27 μM). The results indicate that in case of α(W463F)3β(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent Km of ATPase activity does not represent the ATP binding to a catalytic site. In case of α(K175A/T176A/W463F)3β(Y341W)3γ, the Km of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α(K175A/T176A/W463F)3β(Y341W)3γ was rather linear compared with that of α(W463F)3β(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F1-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed Km's of 100-300 μM and 1-30 μM range for wild-type F1-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively. 相似文献
9.
10.
Mitochondrial ATP synthase (F1F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F1F0-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F1F0-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested. 相似文献
11.
Konno H Suzuki T Bald D Yoshida M Hisabori T 《Biochemical and biophysical research communications》2004,318(1):17-24
To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity. 相似文献
12.
Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF1). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF1 is due to the reduction of the enzyme in vivo in the light. 相似文献
13.
A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF1) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF1 is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg2+ -dependent ATPase activity of the isolated CF1. 相似文献
14.
15.
Temperature-sensitive reaction intermediate of F1-ATPase 总被引:1,自引:0,他引:1
F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step. 相似文献
16.
Why Is the Mechanical Efficiency of F1-ATPase So High? 总被引:2,自引:0,他引:2
The experimentally measured mechanical efficiency of the F1-ATPase under viscous loading is nearly 100%, far higher than any other hydrolysis-driven molecular motor (Yasuda et al., 1998). Here we give a molecular explanation for this remarkable property. 相似文献
17.
Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF1). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF1 is due to the reduction of the enzyme in vivo in the light. 相似文献
18.
Sara Weiss Richard E. McCarty Zippora Gromet-Elhanan 《Journal of bioenergetics and biomembranes》1994,26(5):573-581
SolubilizedRhodospirillum rubrum RrF1-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF1 with Mg2+-ATP or Mg2+-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF1. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF1 has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F1-ATPases have fasterk
off constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg2+ than Ca2+-ATPase activity observed with native RrF1, as with -depleted, activated CF1. An almost complete conversion of both RrF1 and CF1 from Ca2+- to Mg2+-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF1 and CF1 divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg2+ ions. The RrF1-ATPase activity is 30-fold more sensitive than CF1 to efrapeptin, and completely resistant to either inhibition or stimulation by the CF1 effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF1 a low-affinity, stimulatory site for tentoxin.Abbreviations: CF1, EcF1, MF1, and TF1, the soluble F1-ATPase from chloroplasts, PE. coli, mitochondria,R. rubrum, and the thermophilic bacterium PS3, respectively: AMP-PNP, adenylyl-, -imidodiphosphate; CMC, critical micellar concentration; DTT, dithiothreitol, LDAO, lauryl dimethylamine oxide.Dedicated to Professor Achim Trebst in honor of this 65th birthday. 相似文献
19.
A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C. 相似文献
20.
The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites. 相似文献