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1.
(1) Trisbathophenanthroline-Fe 2+(BPh 3Fe 2+) alters the hyperbolic relationship between concentration of ATP and reaction velocity of F 1-ATPase to sigmoidal, with a simultaneous decrease in maximal velocity. (2) BPh 3Fe 2+ binds to the β-subunit of F 1 and competes with the binding of aurovertin. The reversal of this effect by uncouplers in enhanced by ADP and diminished by ATP. BPh 3Fe 2+ also changes the hyperbolic concentration dependence of aurovertin binding to sigmoidal. (3) BPh 3Fe 2+ stabilizes F 1 against cold inactivation and cold dissociation in an uncoupler-reversible manner. (4) BPh 3Fe 2+ efficiently protects F 1 against the light-induced inactivation occurring in the presence of Rose Bengal, and the effect is reversed by uncouplers. (5) The results are discussed in relation to the reaction mechanism of F 1-ATPase and other enzymes catalyzing the reversible hydrolysis of pyrophosphate bonds. 相似文献
2.
(1) Certain metal chelates of 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline, BPh) are potent inhibitors of soluble mitochondrial F1-ATPase. (2) The BPh-metal chelate inhibition of soluble mitochondrial F1-ATPase is relieved by uncouplers of oxidative phosphorylation. (3) The uncouplers appear to interact directly with the inhibitory chelates, forming stoichiometric adducts. (4) A complex between F1 and bPh3Fe2+, containing 3 mol BPh3Fe2+/mol F1, has been isolated. The enzymically inactive F1-BPh3Fe2+ complex binds uncouplers, yielding an enzymically active F1-BPh3Fe2+-uncoupler complex. 相似文献
3.
The pea cotyledon mitochondrial F 1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF 1-ATPase. The molecular weights of pea mitochondrial F 1-ATPase and pea chloroplast CF 1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F 1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF 1-ATPase. The purified mitochondrial F 1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF 1 and F 1, the mitochondrial enzyme stimulated ATP formation in CF 1-depleted pea chloroplast membranes. Thus, the mitochondrial F 1 was able to substitute functionally for the chloroplast CF 1 in reconstituting photophosphorylation. 相似文献
4.
It has been suggested that the F 1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin 1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin 1–7 weakly binds to the rat F 1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin 1–7 binding to the F 1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin 1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin 1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F 1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F 1-ATPase complex. Western blot analysis showed the F 1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F 1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin 1–7 bind to distinct sites on the protein. 相似文献
5.
1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F 1) as the triphosphate, either in the absence or presence of Mg 2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the and 2 mol to the β subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1. 相似文献
6.
Submitochondrial particles were prepared from pea cotyledon mitochondria by sonication in a medium containing 5 mM MgCl 2. The resulting particles (Mg 2+-submitochondrial particles) catalyzed oxidative phosphorylation at the rate of 100–200 nmol ATP formed / min per mg protein. Treatment of Mg 2+-submitochondrial particles with 3.0 M urea resulted in a preparation of highly resolved particles with low ATPase activity and no capacity for oxidative phosphorylation. However, the resulting membranes were not capable of reconstitution of oxidative posphorylation with the purified mitochondrial F 1-ATPase. Urea particles capable of reconstitution of oxidative phosphorylation could be prepared by extracting Mg 2+-submitochondrial particles with concentrations of urea ranging from 1.7 to 2.0 M. We have used 1.9 M urea for large-scale preparation of urea particles that could be stored in liquid nitrogen without any loss of reconstitution capacity. The residual oxidative phosphorylation rate of these particles was 6–8 nmol ATP / min per mg protein and this rate could increase to 60–70 nmol ATP / min per mg protein on incubation with saturating amounts of purified mitochondrial F 1-ATPase. In contrast to the mitochondrial F 1, purified activated pea chloroplast CF 1 was unable to stimulate ATP synthesis in 1.9 M urea particles. 相似文献
7.
The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F 1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H 2O 2 and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F 1. 相似文献
8.
Synthesis of enzyme-bound ATP was demonstrated with purified TF 1 (F 1-ATPase from thermophilic bacterium PS3) from medium inorganic phosphate (P i) and enzyme-bound ADP in the presence of organic solvents such as dioxane, ethanol, dimethylformamide, methanol, acetone, acetonitrile or ethyleneglycol. The optimal concentrations of dimethylformamide, ethanol or methanol were 50%, 30% and 40% and the half-maximal concentrations of P i were 13 mM, 20 mM and 18 mM, respectively. Thus it is evident that the effect of dimethylsulfoxide on TF 1 to form enzyme-bound ATP [8] is not due to a specific interaction between dimethylsulfoxide and the enzyme, but to a decrease in polarity of the medium. In the presence of methanol, the dependence of ATP synthesis on various divalent metal ions was compared to that for the ATP-hydrolyzing activity and the ATP-driven proton-translocating activity of TF 1. While Mn 2+, Co 2+, Zn 2+ and Cd 2+ are as effective as Mg 2+ for the ATP-hydrolyzing activity of TF 1, Zn 2+ and Cd 2+ are either less or not effective for proton translocation and for ATP synthesis. This result appears to be consistent with the idea that the TF 1-ATP complex formed in organic solvents represents one of the intermediates in the reaction sequences of ATP synthesis by H +-ATPase using the proton gradient. 相似文献
9.
以晋西黄土区典型的苹果-玉米间作系统为研究对象,设置了双因素三水平水肥耦合试验,分析不同水肥调控措施下玉米灌浆期穗位叶光合生理特性.本试验根据玉米及苹果适宜的水分和养分条件设置9(3×3)个处理(W 1F 1、W 2F 1、W 3F 1、W 1F 2、W 2F 2、W 3F 2、W 1F 3、W 2F 3、W 3F 3),设置的3个灌溉水平为:田间持水量(Fc)的50%(W 1)、65%(W 2)和85%(W 3), 3个施肥量水平为:N 289 kg·hm -2+ P 2O 5118 kg·hm -2+ K 2O 118 kg·hm -2(F 1)、N 412.4 kg·hm -2 +P 2O 5168.8 kg·hm -2 +K 2O 168.8 kg·hm -2(F 2)、N 537 kg·hm -2 + P 2O 5 219 kg·hm -2 +K 2O 219 kg·hm -2(F 3),另设一组无水肥补给的空白对照(CK).结果表明: 不同水肥调控方式对光合指标日变化趋势无明显影响,但水肥补给可提高作物净光合速率( Pn)的峰值,降低作物日水分利用效率(WUE)最大值,延长气孔开放时间,影响胞间CO 2浓度( Ci)最低值的出现及维持时间;各处理光合作用的限制因素均为非气孔因素.蒸腾速率( Tr)、气孔导度( gs)均与距树行距离呈极显著负相关( P<0.01),水分利用效率则与距树行距离呈显著正相关( P<0.05);距树行距离平均每增加1 m, Tr可减少0.56~1.41 mmol·m -2·s -1, gs可减少0.028~0.093 mol·m -2·s -1,WUE可增加0.08~1.00 μmol·mmol -1.灌水施肥可以显著提高净光合速率、蒸腾速率、气孔导度日均值;降低水分利用效率的日均值;W 3F 1拥有最高的净光合速率日均值(10.64 μmol·m -2·s -1)、水分利用效率日均值(3.05 μmol·mmol -1)、气孔导度日均值(0.295 mol·m -2·s -1)以及较低的蒸腾速率日均值(4.32 mmol·m -2·s -1).多元回归分析结果显示,在拔节-灌浆期内,灌水总量为1300 m 3·hm -2、施肥总量为525 kg·hm -2时,作物净光合速率最大,理论值为10.32 μmol·m -2·s -1.因此,W 3F 1为最利于间作系统作物光合效率改善的水肥调控模式. 相似文献
10.
The protein IF 1 is a natural inhibitor of the mitochondrial F oF 1-ATPase. Many investigators have been prompted to identify the shortest segment of IF 1, retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF 1-(42–58) and IF 1-(22–46) is correlated to their structure and conformational plasticity determined by CD and [ 1H]-NMR spectroscopy. Among all the IF 1 segments tested, IF 1-(42–58) exerts the most potent, pH and temperature dependent activity on the F oF 1 complex. The results suggest that, due to its flexible structure, it can fold in helical and/or β-spiral arrangements that favor the binding to the F oF 1 complex, where the native IF 1 binds. IF 1-(22–46), instead, as it adopts a rigid -helical conformation, it inhibits ATP hydrolysis only in the soluble F 1 moiety. 相似文献
11.
It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F 1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F 1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F 1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F 1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F 1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F 1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F 1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan. 相似文献
12.
The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50-200 μM) causes dose-dependent inhibition of Na +, K +-ATPase activity of rat brain crude synaptosomal-mitochondrial fraction during in vitro incubation up to 2 h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na +, K +-ATPase. Under similar conditions of incubation, DA (200 μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal-mitochondrial proteins and the phenomenon is also prevented by glutathione (5 mM) or cysteine (5 mM). The available data imply that the inactivation of Na +, K +-ATPase in this system involves both H 2O 2 and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na +, K +-ATPase from DA-mediated damage. The inactivation of neuronal Na +, K +-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease. 相似文献
13.
Interaction of mitochondrial F 1-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [ 14C]nucleotides in F 1-ATPase during steady-state [ 14C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein. 相似文献
14.
Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB −), a penetrating anion probe, was used. The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB− uptake by liposomes. The PCB− response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c. Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F1and Fc, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB− uptake. The latter effect was prevented and reversed by oligomycin and uncouplers. The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase). 相似文献
15.
Small-angle neutron scattering experiments were performed in dilute aqueous solutions of chloroplast F 1-ATPase. By contrast variation in 1H 2O/ 2H 2O mixtures and when using different concentrations of glycerol in 2H 2O, structural information on the spatial distribution of dry protein and water was obtained. The maximum distance within latent and active CF 1 was 12 nm. the shape of CF 1 was globular. The total volume of CF 1 was 900 nm 3, and its dry volume (excluding the volume of one water molecule per two exchangeable hydrogen atoms) was 400 nm 3. A volume of 670 nm 3 was inaccessible to glycerol at low glycerol concentrations (less than 25%). At higher concentrations (up to 50%) a volume of 460 nm 3 was excluded to glycerol. Within the resolution of our experiment (1.6 nm) there was no evidence for particular water-rich regions or of secluded water spaces or any particular places for glycerol exchange. Upon thiol activation of the latent enzyme only small changes in structure were detectable just at the limits of the experimental error. They suggest an enhancement of the surface roughness. 相似文献
16.
Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F 0 portion of the F 0F 1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F 0F 1-ATPase complex and these plasmids were used to transform strain AN727 ( uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F 1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F 0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62–69). 相似文献
17.
水资源缺乏和过量施肥影响着干旱半干旱盐渍化地区农业的发展.研究不同灌溉和施肥量对土壤水盐分布和青贮玉米产量的影响,可为该区确定适宜的灌溉和施肥量提供依据.试验于2015和2016年在大同盆地的盐渍化农田进行,设3种灌溉水平:土壤水分上限分别为田间持水率( θf)的100%(W 1)、90%(W 2)和80%(W 3),根据各处理灌溉前的土壤平均实际含水率计算灌水量;2015年设4种施肥水平:900(F 1)、750(F 2)、600(F 3)和450 kg·hm -2(F 4),2016年设F 1、F 2和F 3共3种.试验用化肥为缓释复合肥,总养分含量48%,其中N:P 2O 5:K 2O的比例为30:12:6.结果表明: 土壤表层电导率随施肥量的增加而增大,施肥水平对平均电导率(EC)和含水率的影响在0~10 cm土层显著,与F 1相比,F 2的0~10 cm土层平均EC在2015年和2016年分别降低25.6%~42.7%和6.4%~7.7%.20~80 cm土层的水分含量随施肥量的增加而降低,与F 1相比,2015年F 2、F 3和F 4处理20~80 cm土层平均土壤含水率分别增加5.9%、16.7%和16.7%,2016年F 2和F 3分别增加13.3%和16.7%.产量在两年中均表现为F 1和F 2高于F 3和F 4,W 3低于W 1和W 2; F 1和F 2的产量差异不明显;与W 1相比,W 2的产量减少低于15 %.因此,施复合肥600~750 kg·hm -2(氮肥含量180~270 kg·hm -2),且灌溉水平为W 1和W 2时,可以保证该地区盐渍化土壤种植玉米获得较高的产量,并且不会造成根系层的盐分积累. 相似文献
18.
土壤线虫是指示土壤健康的典型生物之一,为了揭示氮肥减施对土壤健康的影响,以冬小麦土壤为对象,研究了氮肥减施和配施有机肥对拔节期冬小麦土壤线虫群落结构的影响。共设置了6个施肥处理:CF(315 kg N·hm -2,常规施肥量)、N 240(240 kg N·hm -2)、N 210(210 kg N·hm -2)、N 180(180 kg N·hm -2)、F 150(180 kg N·hm -2+150 kg·hm -2黄腐酸)、F 225(180 kg N·hm -2+225 kg·hm -2黄腐酸)。结果表明: 1)氮肥减施会降低土壤线虫数量,降幅为15.3%~68.5%;2)各处理均以原杆属为优势属(19.6%~50.4%)。氮肥减施提高了食真菌类、植食类和捕食/杂食类线虫的丰度,食细菌类线虫的丰度先降低后升高。配施有机肥后,食细菌类和食真菌类线虫的丰度降低,植食类和捕食/杂食类线虫丰度升高;3)N 240和F 225处理分别使线虫多样性指数 H提高了48.1%和58.5%。N 240处理线虫成熟度指数 MI最高(1.95)。N 180处理线虫结构指数 SI最低(43.33),配施有机肥的F 225线虫结构指数 SI达到62.72,但线虫富集指数 EI最低(80.82)。说明减施氮肥并配施有机肥可以提高土壤线虫的多样性,使食物网向复杂稳定方向发展,对农田土壤生态系统有积极意义。 相似文献
19.
Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP +) on the oxygen consumption, ATP production, H 2O 2 production, and mitochondrial NADH-CoQ 1 reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP + had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP + produced a concentration-dependent decrease in H 2O 2 production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP + caused no loss of complex I activity after washing of MPP + from the mitochondrial membranes. These data are consistent with MPP + initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H 2O 2 by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H 2O 2 in the presence of Cu 2+ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP + is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect. 相似文献
20.
Reactions of cis-diamminedichloroplatinum(II) with phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and methylenediphosphonic acid (MDP) yield various phosphonatoplatinum(II) chelates which were characterized by phosphorus-31 NMR spectroscopy. The P-31 resonances for the chelates appear at 6–12 ppm downfield as compared to the uncomplexed ligands. All complexes exhibit monoprotic acidic behavior in the pH range 2–10. The chemical shift-pH profiles yielded acidity constants, 1.0 × 10 −4, 1.5 × 10 −4, and 1.3 × 10 −6 M −1, for the PFA, PAA, and MDP chelates. In addition to the monomeric chelate, MDP formed a bridged diplatinum(II,II) complex when it reacted with cis-Pt (NH 3) 2(H 2O) 22+. The P-31 resonance for this binuclear complex appears at 22 ppm downfield from the unreacted ligand. Rate data for the complexation reactions of the phosphonate ligands with the dichloroplatinum complex are consistent with a mechanism in which a monodentate complex is formed initially through rate-limiting aquation process of the platinum complex, followed by a rapid chelation. For the PFA and PAA complexes, initial binding sites are the carboxylato oxygens. Implications of the various binding modes of the phosphonates in relationship to their antiviral activities are discussed. 相似文献
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