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1.
The coupled mitochondria of maize seedlings are the site of electron-transport-dependent synthesis of inorganic pyrophosphate. The inorganic-pyrophosphate synthesis depends on the presence of Mg 2+ and exogenous phosphate; it is inhibited by electron transport inhibitor, uncoupler and by inorganic pyrophosphatase inhibitors (methylene diphosphonate, NaF, Ca 2+). 相似文献
2.
The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl 2 and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers. 相似文献
3.
Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores. 相似文献
4.
The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-P i exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-P i exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-P i exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD + reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling. 相似文献
5.
Chromatium vinosum chromatophores contain an energy-linked pyrophosphatase that is insensitive to oligomycin and dicyclohexylcarbodiimide. Pyrophosphate hydrolysis produces a carotenoid band-shift similar to that resulting from illumination. The carotenoid band-shift can also be produced by a K + diffusion potential (interior positive) and the magnitude of the band shift is proportional to the membrane potential over at least a 100-fold variation in K + concentration. The light-induced carotenoid band-shift in whole cells is identical to that seen in chromatophores but K + diffusion potentials (interior positive) produce a mirror image of the light-induced band-shift. These results are interpreted in terms of chromatophores being inside-out vesicles. 相似文献
6.
The effect of selected inhibitors on the thrombin-stimulated burst and the basal oxygen consumption of washed human platelets were investigated and compared with inhibition of the release reaction. Cyanide (0.2 mM) caused complete inhibition of the basal respiration, but only 15% inhibition of the thrombin-stimulated burst of oxygen consumption. Similar differential inhibitory effects were observed with oligomycin, antimycin, rotenone and N-ethylmaleimide. Prostaglandin E 1 (0.03 mM) and acetylsalicylic acid (0.8 mM) had little effect on basal respiration, but inhibited the thrombin-stimulated burst of oxygen consumption. N-Ethylmaleimide (0.4 mM) inhibited the release of calcium from platelets by 90%, while prostaglandin E 1, acetylsalicylic acid and the above mitochondrial inhibitors caused no more than 30% inhibition of the release reaction. Our results provide evidence that basal respiration and a portion of the thrombin-stimulated burst of oxygen consumption are involved in respiratory chain phosphorylation, and that this component of the thrombin-stimulated burst may be coupled to the maintenance of the release reaction. 相似文献
7.
Photosynthetic formation of inorganic pyrophosphate (PP i) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP i synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP i synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP i synthesis much less. Even at low rates of electron transport a significant rate of PP i synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PP i synthesis is inhibited. It is shown that PP i synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions. 相似文献
8.
The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P i exchange reactions of R. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine. 相似文献
9.
Treatment of a purified (Na + + K +)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na + + K +)-ATPase reaction inhibited more than the K +-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na + + K +)-ATPase reaction but not the K +-phosphatase reaction; however, treatment with digitonin made the K +-phosphatase reaction almost as sensitive to oligomycin as the (Na + + K +)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K +-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K +-phosphatase reaction more than the (Na + + K +)-ATPase reaction). Both digitonin and Triton markedly increased the K0.5 for K + as activator of the K +-phosphatase reaction, with little effect on the K0.5 for K + as activator of the (Na + + K +)-ATPase reaction. In contrast, increasing the K0.5 for K + in the K +-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K +-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E 1 conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E 2 state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K0.5 for K + in the K +-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na + plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na + plus nucleotide. 相似文献
10.
A simple method for measuring PPi at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PPi. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PPi in urine is demonstrated. 相似文献
11.
The mechanism of light-induced O 2 uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O 2 uptake by the extracted chromatophores and stimulated the O 2 uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O 2. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O 2 upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl- p-phenylenediamine (Me 4Ph(NH 2) 2) or N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB ?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD + or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O 2, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me 4Ph(NH 2) 2 to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me 4Ph(NH 2) 2 and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me 4Ph(NH 2) 2 and TMPD are different. 相似文献
12.
DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. 相似文献
13.
An ADP-ATP exchange reaction was studied in NaI-treated microsomes from rat brain. At low concentrations of MgCl 2 a nucleotide exchange reaction, elicited in the presence of Na +, had an absolute specificity for ATP. The reaction was stimulated by oligomycin and inhibited by ouabain and EDTA. It is probable that this Na +-stimulated ADP-ATP exchange reaction is a component of the Na-K ATPase system. 相似文献
14.
Alkaline inorganic pyrophosphatase and Mg-ATPase are localized within the mitoplast of maize seeding mitochondria. NaF inhibited the PPase activity, whereas oligomycin and dicyclohexylcarbodiimide inhibited the Mg-ATPase activity. The mitoplast preparation synthesized PPi from Pi under conditions excluding hydrolysis of endogenous ATP. PPi synthesis was inhibited by ADP, antimycin A, NaCN and 2,4- dinitrophenol but not by oligomycin. It is suggested that PPi synthesis in the maize seedling mitochondria proceeds at the expense of the energy of electron transport chain and is independent of the ATP synthesis. 相似文献
15.
In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PP i) in three phototrophic bacteria. Formation of PP i was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PP i synthesis in Rps. viridis was 0.15 mol PP i formed/(min*mol Bacteriochlorophyll) at 23°C. The synthesis of PP i was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PP i synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PP i after short light flashes was also studied. The yield of PP i after a single 1 ms flash was equivalent to approximately 1 mol PP i/500 mol Bacteriochlorophyll. In Rps. viridis chromatophores, PP i was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.Abbreviations BChl
Bacteriochlorophyll
- F 0F 1-ATPase
Membrane bound proton translocating ATP synthase
- FCCP
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone
- H +-PPase
Membrane bound proton translocating PP i synthase
- TPP +
Tetraphenyl phosphonium ion
- TPB -
Tetraphenyl boron ion
-
Transmembrane electrical potential difference 相似文献
16.
The aerobic photooxidations of reduced 2,6-dichlorophenolindophenol and of reaction-center bacteriochlorophyll ( P-870) have been investigated in membrane vesicles (chromatophores) isolated from a non-phototrophic Rhodospirillum rubrum strain. In aerobic suspensions of wild-type chromatophores, continuous light elicits an increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which reach steady-state values shortly after the onset of illumination. In contrast, light induces in mutant suspensions a transient increase of the levels of 2,6-dichlorophenolindophenol and of oxidized P-870, which fall to low steady-state values within a few seconds. These observations suggest that the mutation has altered a redox constituent located on the low-potential side of the photochemical reaction center, between a pool of acceptors and oxygen.Since endogenous cyclic photophosphorylation is catalyzed by mutant chromatophores at normal rates, it appears that the constituent altered by the mutation does not belong to the cyclic electron-transfer chain responsible for photophosphorylation. However, the system which mediates the aerobic photooxidations and the cyclic system are not completely independent: endogenous photophosphorylation is inhibited by oxygen in wild-type chromatophores but not in mutant chromatophores; in addition, the inhibitor of cyclic electron flow, 2-heptyl-4-hydroxyquinoline- N-oxide, enhances the aerobic photooxidation of reduced 2,6-dichlorophenolindophenol by chromatophores from both strains.These results support a tentative branched model for light-driven electron transfer. In that model, the constituent altered in the mutant strain is located in a side electron-transfer chain which connects the cyclic acceptors to oxygen. 相似文献
17.
A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used. 相似文献
18.
The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O 2. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K 4Fe(CN) 6, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c2 from the heavy chromatophores caused a decrease in the light-induced O 2 uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c2 from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c2 level which faces the outside medium. However, cytochrome c2 is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c2 in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O 2 whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O 2 consumed is 4, which means that the end product of O 2 reduction is H 2O. 相似文献
19.
An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an
2
2 oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg 2+ was required for activity. The pH optimum was 8 at 1 mM PP
i
and 5 mM Mg 2+. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent K
m for PP
i
of 0.1 mM in the presence of 5 mM Mg 2+. The V
max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K
cat of 1400 per second.The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedyre were applied.Abbreviation PMSF
phenylmethylsulfonyl fluoride 相似文献
20.
The particulate fraction of Rhodopseudomonas viridis when supplied with succinate catalyses the reduction of NAD + by light; this reaction is inhibited by uncouplers of oxidative phosphorylation but not by oligomycin. Formation of NADH takes place in the dark when ATP or PP i is supplied. Both light and dark reactions are inhibited by valinomycin and nigericin, when added together, but not by either separately. NADH formation in R. viridis appears to take place by an energy-dependent reversal of electron flow and energy may be conserved in the form of a membrane potential. The addition of ATP caused the oxidation of both C553 and C558 in chromatophores; carbonylcyanide p-trifluoromethoxyphenylhydrazone and oligomycin abolished this oxidation. The NAD+ and NADH concentrations at equilibrium in the light-dependent reaction were determined and the oxidation-reduction potential of this couple calculated. From this value it was calculated that under these experimental conditions the energy requirement to form NADH from the succinate/fumarate couple at Eh = o V was 9.4 kcal. Particles of R. viridis contained an active transhydrogenase, driven by either light or ATP, that was sensitive to uncouplers of oxidative phosphorylation; the light-driven reaction was insensitive to oligomycin and was inhibited by antimycin A and 2-heptyl-4-hydroxyquinone-N-oxide. R. viridis did not grow aerobically but particles contained NADH oxidase activity that was cyanide sensitive. There was no spectroscopic evidence for cytochromes of the b-type in reduced-minus-oxidised spectra of particles or in pyridine haemochrome spectra of whole cells. 相似文献
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