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1.
M.P. Roisin  J.P. Henry 《BBA》1982,681(2):292-299
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   

2.
Bovine heart submitochondrial particles depleted of F1 by treatment with urea (‘F1-depleted particles’) were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH+4, K+, Rb+, Cs+, Na+ and Li+ promoted reconstitution maximally at 40–74 mM, guanidinium+ and Tris+ at 20–30 mM, and Ca2+ and Mg2+ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.  相似文献   

3.
Coupling factor 6 (F6) and mitochondrial ATPase inhibitor were isolated from the rutamycin-sensitive ATPase complex of bovine heart mitochondria by heating and fractionation with ethanol. F6 appeared in acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and urea as a single band corresponding to a molecular weight of 8,000. This protein which is required for the 32Pi-ATP exchange in submitochondrial particles treated with silicotungstate was very sensitive to trypsin.  相似文献   

4.
Three proton pumps,morphology and movements   总被引:3,自引:0,他引:3  
The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 µg per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.Abbreviations SMP submitochondrial vesicles prepared from bovine heart mitochondria exposed to sonic oscillation in the presence of pyrophosphate - F1 the water-soluble coupling factor of the mitochondrial ATPase complex - CF1 the water-soluble coupling factor of the chloroplast ATPase complex - ASU vesicles submitochondrial vesicles prepared from bovine heart mitochondria disrupted by sonic oscillation in ammonia, then passed through Sephadex and treated with urea - OSCP oligomycin sensitivity-conferring protein - Mega 8, 9, and 10 for octoylnanoyl, and decanoyl-N-methylglucamide - 1799 bis-(hexafluoroacetonyl)acetone - PMS N-methylphenazonium methosulfate  相似文献   

5.
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   

6.
Ronald S. Kaplan  P.S. Coleman 《BBA》1978,501(2):269-274
1. The use of 1,N6-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F1-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F1-ATPase displays similar Km values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.  相似文献   

7.
Beef heart mitochondrial protein factor FB [Higashiyama etal, Biochemistry 14, 4117–4121 (1975)] was purified and its properties were compared with those of coupling factor B. Both proteins stimulated ATP-driven NAD+ reduction in ammonia and EDTA-treated (AE-) submitochondrial particles, but the extent of stimulation (maximum activity of particles) was very low with FB. FB was found to be ineffective in stimulating Pi-ATP exchange in either AE-particles or reconstituted oligomycin-sensitive ATPase vesicles. Furthermore, FB failed to stimulate ATP-driven NAD+ reduction activity of AE-particles in the presence of saturating amounts of dithiothreitol (DTT). DTT alone stimulates the particle activity extensively as reported earlier. Rabbit antiserum to FB did not show a precipitin band with purified Factor B, nor did the antibody inhibit Factor B stimulated activity of the AE-particles. The data suggest that FB and Factor B are two different molecular species with different functions and fail to provide evidence that FB is a coupling factor.  相似文献   

8.
The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.  相似文献   

9.
The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F 1, Mg++, phospholipids, and Fc. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c 1, cytochrome c, cytochrome oxidase, phospholipids and Q 10. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.  相似文献   

10.
Mitochondrial H+-ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a “membrane” (NaBr-F0) and a “soluble” fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of p, 8, and e subunits of the F, ATPase and largely devoid of α and γ subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and P1-ATP exchange activities. The addition of F1 (400 μg · mg?1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pf-ATP exchange and H+-pumping activities require coupling factor B in addition to FpATPase. The oligomycin-sensitive ATPase and 32P1ATP exchange activities in reconstituted Fr F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1F0 preparations rather than to sodium bromide treatment itself.

The H+-ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35–37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler-and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and P1-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial Fo is not known.

The F0 preparations from bovine heart reported so far have been derived from H+-ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37). The ATPase and P1-ATP exchange activity of the preparations so obtained are low, dependent upon additional phospholipids and coupling factors; they show altered sensitivity to energy transfer inhibitors as compared to submitochondrial particles from the heavy layer of the mitochondria or ETPh (1. 2, 12, 14, 29, 33). Recently, lysolecithin has been successfully employed to extract highly active H+-ATPase from beef (17, 19, 28) and pig (24) heart mitochondria. The beef heart H+-ATPase preparation has the same ratio of ATPase to PrATP exchange activity and apparently the same sensitivity to energy transfer inhibitors as submitochondrial particles (17). The present communication describes resolution of this F1-F0 preparation using sodium bromide (NaBr) and reconstitution of ATPase and Pr ATP exchange activities. The NaBr-F0 prepared from this preparation shows no dependence on lipids, and the same or increased sensitivity to energy transfer inhibitors when reconstituted with F1-ATPase. Furthermore, F1 ATPase activity does not decrease on binding of F1 to NaBr-F0, even though the reconstituted ATPase activity is 99% sensitive to oligomy-cin and dicyclohexylcarbodiimide. These properties are in contrast to the properties of F0 reported by other workers (12, 14).  相似文献   

11.
Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg2+ and in the presence of Mg2+ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl->HCO 3 - >Br->malate>acetate>SO 4 2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH 4 + Na+>K+). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na+ and K+. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in Vmax rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO3 but other inhibitors tested (azide, oligomycin, orthovanadate, K3[Cr(oxalate)6] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATPF free ATP - ATPT total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - Km apparent Michaelis constant - MgATP complex of Mg2+ and ATP - Mg F 2+ free Mg2+ - Mg T 2 total Mg2+ - MES 2-(N-Morpholino)ethanesulphonic acid - Na2EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - Pi inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - Vmax maximum velocity  相似文献   

12.
The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F1-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca2+-uptake of mitochondria energized both by ATP or succinate without affecting the Ca2+-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the Pi and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/  相似文献   

13.
Summary A vanadate-sensitive H+-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H+-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H+ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H+ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na3VO4) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aK i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.  相似文献   

14.
Leaves ofNerium oleander L. plants, which had been previously kept in a shaded glasshouse for at least two months, were fed 1 mM dithiothreitol (DTT) through their petioles, either for 12h in darkness (overnight) or for 2h in low light (28 μmol photons·m−2·s−1), in each case followed by a 3-h exposure to high light (1260 μmol photons·m−2·s−1). During exposure to high light, violaxanthin became converted to zeaxanthin in control leaves, to which water had been fed, whereas zeaxanthin did not accumulate in leaves treated with DTT. Total carbon gain was not reduced by DTT during the photoinhibitory treatment. Exposure to high light led to a decrease in the photochemical efficiency of photosystem II, measured as the ratio of variable over maximum fluorescence emission,F v/F M, at both 298 K and 77K. The decrease was much more pronounced in the presence of DTT, mainly owing to a sustained increase in the instantaneous fluorescence,F o. By contrast, in the control leaves,F o determined immediately after the high-light treatment showed a transient decrease below theF o value obtained before the onset of the photoinhibitory treatment (i.e. after 12 h dark adaptation), followed by a rapid return (within seconds) to this original level ofF o during the following recovery period in darkness. Incubation of leaves with DTT led to large, sustained decreases in the photon-use efficiency of photosynthetic O2 evolution by bright light, whilst the capacity of photosynthetic O2 evolution at light and CO2 saturation was less affected. In the control leaves, only small reductions in the photon yield and in the photosynthetic capacity were observed. These findings are consistent with previous suggestions that zeaxanthin, formed in the xanthophyll cycle by de-epoxidation of violaxanthin, is involved in protecting the photosynthetic apparatus against the adverse effects of excessive light.  相似文献   

15.
The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F1Fo-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K0.5=0.27 mM) and low (K0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg2+ saturating condition (high affinity site, K0.5=0.10 mM, and low affinity site, K0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.  相似文献   

16.
Acid tolerance mediated by membrane ATPases in Lactobacillus acidophilus   总被引:1,自引:0,他引:1  
The acid tolerance response in Lactobacillus acidophilus CRL 639, induced at pH 4.2 for 15 min, is mediated by the cell membrane F 1-F 0 ATPase. The specific activity of the enzyme was induced 1.6-fold after acid adaptation compared to a non-adapted control. The ATPase was optimal at pH 6 with a K m=0.8 mM and a V max=100 mM.  相似文献   

17.
18.
Phosphatase activities were measured in preparations of vacuoles isolated from storage roots of red beet (Beta vulgaris L.). The vacuoles possessed both acid phosphatase and ATPase activities which could be distinguished by their susceptibility to inhibition by low concentrations of ammonium molybdate [(NH4)6Mo7O24·4H2O]. The acid phosphatase was completely inhibited by 100 M ammonium molybdate but the ATPase was unaffected. The acid phosphatase was a soluble enzyme which hydrolysed a large number of phosphate esters and had a pH optimum of 5.5. In contrast, the ATPase was partially membrane-bound, had a pH optimum of 8.0 and hydrolysed ATP preferentially, although it was also active agianst PPi, GTP and GDP. At pH 8.0 both the ATPase and PPase activities were Mg2+-dependent and were further stimulated by KCl. The ATPase and PPase activities at pH 8.0 may be different enzymes. The recovery and purification of the ATPase during vacuole isolation were determined. The results indicate that the Mg2+-dependent, KCl-stimulated ATPase activity is not exclusively associated with vacuoles.Abbreviations BSA bovine serum albumen - MES 2-(N-Morpholino)ethanesulphonic acid - MOPS 3-(N-Morpholino)propanesulphonic acid - Na2EDTA ethylenediaminetetra-acetic acid, disodium salt - Pi inorganic phosphate - PPi inorganic pyrophosphate - PPase inorganic pyrophosphatase - TCA trichloroacetic acid - TES N-tris(hydroxymethyl)methyl-2-amino-ethanesulphonic acid - Tris tris(hydroxymethyl)methylamine  相似文献   

19.
In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF1. Photosynthetic membranes of Spirulina maxima show a Mg2+-dependent ATPase activity of 195±25 mol Pi (mg chl)–1 h–1 after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg2+-dependent ATPase activity of 222±18 mol Pi (mg chl)–1 h–1.Here, we also describe the purification of the soluble coupling factor AF1 of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F1 preparations in regard to its high specific Ca2+-dependent ATPase activity after heat treatment (14.75±1.91 mol Pi (mg prot)–1 min–1) and its room temperature stability.Abbreviations AF1 cyanobacterial coupling factor - DTT dithiothreitol - DEAE cellulose diethylaminoethyl cellulose - DCCD N,N'-dicyclohexylcarbodiimide - EDTA ethylenediamine tetracetic, sodium salt - PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulfate - PMSF phenylmethylsulfonyl fluoride - MV methylviologen - SDS sodium dodecylsulfate - TPSnCl triphenyltin chloride - Tris tris (hydroxymethyl) aminomethane - tricine N Tris (hydroxymethyl) methylglycine - BAEE N-benzoyl-L-arginine ethyl ester  相似文献   

20.
Bacteriorhodopsin-F1·F0 (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F1·F0 is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F1·F0 by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [32P]ATP formation where pyridine is present during first stage illumination.F1·F0 can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.  相似文献   

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