共查询到20条相似文献,搜索用时 31 毫秒
1.
I J Ryrie 《Archives of biochemistry and biophysics》1975,168(2):704-711
A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit ATP-32Pi exchange when combined with phospholipids. The reconstitution was normally carried out by dialysis of an ATPase-phospholipid-bile detergent mixture, but could also be achieved by direct addition of the lipid. Vesicle structures with diameters between 200 and 1500 Å were seen by electron microscopy.The ATP-32Pi exchange was independent of electron transport but sensitive to uncouplers and energy-transfer inhibitors. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. Taken together, the results raise the possibility that the terminal coupling mechanism might still be intact within the ATPase complex. 相似文献
2.
Ivan J. Ryrie 《Journal of cellular biochemistry》1975,3(3):242-247
A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin- and uncoupler-sensitive ATP-32Pi exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase-phospholipid-cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 Å were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex. 相似文献
3.
(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-32Pj exchange. The vesicles so formed exhibit proton uptake upon addition of Mg2+-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-32Pi exchange by valinomycin + K+ reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys. 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K+ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-32Pi exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [32P]ATP formation from ADP and 32Pi is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin. 相似文献
4.
B Blanchy C Godinot D C Gautheron 《Biochemical and biophysical research communications》1978,82(3):776-781
The use of mitoplasts, that is mitochondria devoid of outer membrane oriented as normal mitochondria, and of sonicated vesicles, the membrane of which is inside-out has shown that the thiol groups involved in the process of ATP synthesis are on the matrix face of the mitochondrial membrane: carboxypyridine disulfide (CPDS) a thiol reagent that cannot penetrate across hydrophobic membranes does not inhibit the ATP-32Pi exchange catalyzed by mitoplasts, while 5,5′-dithio--(2-nitrobenzoate), which penetrates more readily, can completely inhibit this exchange. In contrast, both reagents react similarly with inside-out vesicles. The nature of the component of the ATPase-ATP synthase complex to which this thiol group may belong is discussed. 相似文献
5.
In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+-ATPase complex. These effects are caused by nitration of tyrosine residues of H+-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles. H+ conduction in phospholipid vesicles inlaid with F0 is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F0 are critically involved in energy-linked proton translocation in the ATP-ase complex. 相似文献
6.
An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-32PPi exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-32PPi exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined. 相似文献
7.
Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F1-ATPase were major components of the preparation. 相似文献
8.
Summary Resting cells ofStaphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of32Pi (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi anddl-lactate to establish an internal pool of Pi. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi.Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous32PiPi exchange (K
i's of 2.2 and 1.4mm;V
max's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K
i=27 m) over G3P (K
i=1.3mm) and Pi (K
i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that32PiPi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, Pi-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport. 相似文献
9.
We previously demonstrated inhibition of Na+-dependent 32Pi transport in canine renal brush-border membranes in association with NAD+-induced ADP ribosylation of membrane protein(s) and postulated that NAD+ inhibits Pi transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD+ resulted in release of Pi which was prevented by EDTA. It was proposed that NAD+-mediated inhibition of 32Pi transport might occur through this mechanism. To determine whether NAD+ inhibited 32Pi transport by a mechanism other than or in addition to release of Pi, we compared Na+-dependent 32Pi counterflow in brush-border membrane equilibrated with Pi or with Pi generated from NAD+. Release of Pi from NAD+ incubated with brush-border membrane was confirmed. The increased uptake of 32Pi which was demonstrated in brush-border membrane equilibrated with Pi was not measured when intravesicular Pi was generated from a concentration of NAD+ which effected ADP-ribosylation of brush border membranes (100 μM NAD+). In contrast, increased uptake of 32Pi was demonstrated when intravesicular Pi was generated from 1 μM NAD+ which did not effect ADP ribosylation. Mg2+-dependent ADP ribosylation of brush-border membrane incubated with NAD+ was demonstrated which persisted during the time interval of 32Pi uptake measurements. Our findings are compatible with the hypothesis that NAD+-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of Pi transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg2+ and decreased ADP ribosylation. 相似文献
10.
1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Pi is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation. 相似文献
11.
K. Hotta 《Journal of cellular biochemistry》1975,3(4):333-337
Myosin catalyzed exchange between 32Pi and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of 32Pi in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange 32Pi and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of 32Pi incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested. 相似文献
12.
Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi ATP exchange reaction with bound Pi The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers. 相似文献
13.
Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli 总被引:4,自引:0,他引:4
Mohamed A. Marahiel Michael Krause Hans-Jacob Skarpeid 《Molecular & general genetics : MGG》1985,201(2):231-236
Summary The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific d-phenylalanine dependent ATP-32PPi and 2deoxy ATP-32PPi exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector. 相似文献
14.
C.F. Dick A.L.A. Dos-Santos D. Majerowicz L.S. Paes N.L. Giarola K.C. Gondim A. Vieyra J.R. Meyer-Fernandes 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi.Methods
32Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na+, H+ and K+ fluxes were also investigated. The transport capacities of different evolutive forms were compared.Results
Epimastigotes grew significantly more slowly in 2 mM than in 50 mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na+. We found that the parasites express TcPho84, a H+:Pi-symporter, and TcPho89, a Na+:Pi-symporter. Both Pi influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na+-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K+ ionophore) or SCH28028 (inhibitor of (H+ + K+)ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H+ gradient energizes uphill Pi entry and that K+ recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, decreased only the Na+-dependent Pi uptake, indicating that this Na+ pump generates the Na+ gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently.Conclusions
Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na+ or H+/K+ fluxes.General significance
This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms. 相似文献15.
S. Passarella F. Palmieri E. Quagliariello 《Archives of biochemistry and biophysics》1977,180(1):160-168
The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial Pi, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/Pi exchange, but only partially the oxaloacetate/malonate and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetateout/32Piin, whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetateout/[14C]oxoglutaratein exchange. The apparent Km values ranged from 0.6 to 1.2 mm for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mm for the oxaloacetate/Pi exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/Pi exchange competitively and it is a noncompetitive inhibitor of the exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed. 相似文献
16.
In view of the importance of Pi in the control of cell metabolism, it was of interest to study the mechanism and regulation of Pi uptake by ascites tumor cells. For this purpose, the incorporation of 32Pi into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease 32Pi uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 Mr anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases 32Pi incorporation at concentrations as low as 2 × 10?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na+ + K+)-ATPase and its dephosphorylation, stimulates the rate of incorporation of 32Pi into cells and also raises the net inorganic phosphate level. The stimulation of 32Pi incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K+, which is known to stimulate (Na+ + K+)-ATPase and its dephosphorylation, decreases 32Pi incorporation. These observations suggest that anion transport and energy-dependent Na+ and K+ movements may be closely coupled to the intact cell. 相似文献
17.
Oxidative phosphorylation and respiratory control in lysolecithin treated electron transport particles 总被引:2,自引:0,他引:2
D R Hunter H Komai R A Haworth 《Biochemical and biophysical research communications》1974,56(3):647-653
Lysolecithin treatment of electron transport particles (ETP) generated non-vesicular fragments of membrane that can catalyze oxidative phosphorylation. Electron micrographs of ultrathin sections of lysolecithin treated ETP were devoid of circular patterns characteristic of closed vesicular structures. No synergistic uncoupling of oxidative phosphorylation by valinomycin plus nigericin in the presence of K+ was observed in such fragments of membrane, which remained sensitive to classical uncouplers and to oligomycin. Preceding total destruction of closed vesicular structure, lysolecithin caused a drastic alteration in the membrane as evidenced by a greatly diminished effect of the ionophores in releasing respiratory control. 相似文献
18.
《Molecular membrane biology》2013,30(2):203-225
A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the Pi/OH exchange, but not the Pi/Pi exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K+. Both Pi/OH and Pi/Pi exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl. 相似文献
19.
Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein 总被引:2,自引:0,他引:2
Y Kagawa L W Johnson E Racker 《Biochemical and biophysical research communications》1973,50(2):245-251
Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid32Pi-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little32Pi-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [32P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation. 相似文献
20.
A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P]-ATP formed from 32Pi by S1 catalysis and the amount of rapidly formed [32P]-ATP tightly bound to S1, the value of the off constant is approximately 2.8 × 10−4 sec−1 at pH 7.4. The concentration dependencies for Pi ⇌ H18 OH exchange and for 32Pi incorporation into myosin-bound ATP give direct measurements of the dissociation constant of Pi from S1. Both approaches show that the enzyme has a very low affinity for Pi, with an apparent Kd of > 400 mM. Measurement of the average number of water oxygens incorporated into Pi released from ATP by S1-catalyzed hydrolysis in the presence of Mg2+ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca2+-activated hydrolysis, less than one oxygen from water appears in each Pi released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP. 相似文献