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1.
The effect of chemical modifiers of amino acid residues on the proton conductivity of H+-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H+ in ESMP as well as in particles deprived of F1 (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H+ exhibited two first-order phases, both an expression of the H+ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H+ conduction caused by respiratory H+ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H+ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H+ conduction. In USMP, H+ release followed simple first-order kinetics. Oligomycin depressed H+ release, enhanced respiratory H+, and restored the biphasicity of H+ release. Phenylglyoxal and tetranitromethane inhibited H+ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H+ release from USMP, introducing a very rapid phase of H+ release. Addition of soluble F1 to USMP also restored biphasicity of H+ release. A mechanism of proton conduction by F
o
is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H+ conduction, and those modified by butanedione, which retard H+ diffusion. 相似文献
2.
David G. Griffiths Michael J. Pringle James B. Hughes D. Rao Sanadi 《Journal of bioenergetics and biomembranes》1984,16(5-6):465-475
Electron transport particles and purified H+-ATPase (F1-Fo) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series ofN-polymethylene carboxymaleimides (Mal-(CH2)
x
-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive.N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector Fo was 50% blocked by AM10, slightly blocked by AM2 andN-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 Å from the surface are functional in proton translocation through the H+-ATPase. 相似文献
3.
Summary
Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol Pi/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H+ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H+-ATPase. Unusual properties of this enzyme are: (i) theK
m for ATP is around 60 M, considerably lower than in other plasma membrane H+-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K+ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H+-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient. 相似文献
4.
猪心线粒体Fo的纯化、重建及其质子转运功能 总被引:1,自引:0,他引:1
比较了猪心线粒体FoF1-ATPase膜部分Fo的四种纯化方法.结果表明,用NaBr从亚线粒体除去FoF1-ATPase的水溶性部分F1-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的Fo.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的Fo含有b、OSCP(寡霉素敏感授予蛋白)、d、a、e、F6、IF1、A6L和c等9种亚基.用去污剂稀释法将纯化的Fo在脂质体上重建后,重建Fo表现较高的被动转运质子活性.这为在体外深入研究Fo的活性、构象与膜脂的关系,以及Fo与F1-ATPase的组装等提供了很好的实验模型. 相似文献
5.
The proton-motive forces generated in submitochondrial particles by both hydrolysis of ATP and oxidation of succinate have been measured by flow dialysis and compared with the ambient phosphorylation potentials. It is concluded that three H+ are translocated for each ATP molecule hydrolysed or synthesised. By utilising rat liver mitochondria respiring with β-hydroxybutyrate as a new system for regeneration of ATP from ADP and Pi, phosphorylation potentials were clamped at a range of values by using mixtures of particles and mitochondria in various ratios. As the rate of ATP hydrolysis by the particles was lowered, the proton-motive force decreased only slightly except at the very lowest rates, these results paralleling earlier studies on the relation between rate of respiration-driven proton translocation and proton-motive force. 相似文献
6.
《Molecular membrane biology》2013,30(4):309-325
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
7.
Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H+/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H+/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K+ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H+/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K++H+)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach. 相似文献
8.
The bacteriocin butyricin 7423 inhibited the activity of the membrane H+-ATPase (BF0, F1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF0 component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.Non-standard abbreviations H+-ATPase
proton translocating adenosine 5-triphosphatase (EC 3.6.1.3)
- DCCD
dicyclohexylcarbodiimide 相似文献
9.
Yuan Ru Tsai Su Jing Yang Shih Sheng Jiang Shu Jin Ko Shu Hsien Hung Soong Yu Kuo Rong Long Pan 《Journal of Protein Chemistry》1998,17(2):161-172
A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H+-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H+-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V
max and K
m of solubilized vacuolar H+-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg2+-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H+-ATPase by pressure may result from changes in protein–protein interaction. The conformational change of solubilized vacuolar H+-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H+-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit–subunit interaction is crucial for the integrity and the function of plant vacuolar H+-ATPase. It is also suggested that the assembly of the vacuolar H+-ATPase complex is probably not random, but follows a sequestered pathway. 相似文献
10.
Jesper Torb?l Pedersen Janus Falhof Kira Ekberg Morten Jeppe Buch-Pedersen Michael Palmgren 《The Journal of biological chemistry》2015,290(33):20396-20406
The plasma membrane H+-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H+/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H+-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H+-ATPases is labile in the basal state, which may provide an explanation for the low H+/ATP coupling ratio of these pumps in the basal state. 相似文献
11.
The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F0F1-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K
m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K
m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F0F1-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely. 相似文献
12.
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
13.
James Hughes Saroj Joshi Katalin Torok D. Rao Sanadi 《Journal of bioenergetics and biomembranes》1982,14(5-6):287-295
The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400–600 nmol × min–1 × mg–1). This activity is further enhanced (1400–1600 nmol × min–1 × mg–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H+ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. Pi-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP). 相似文献
14.
(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
15.
Proton transport-coupled unisite catalysis was measured with the H+-ATPase from chloroplasts. The reaction was measured in the ATP hydrolysis direction under deenergized conditions and in the ATP synthesis direction under energized conditions. The equilibrium constant of the enzyme does not change upon energization, whereas the dissociation constants of substrates and products change by orders of magnitude. This indicates that the Gibbs free enthalpy derived from proton translocation is used to change binding affinities of substrates and products, and this results in synthesis of free ATP. 相似文献
16.
Chelysheva V. V. Zorinyants S. E. Smolenskaya I. N. Babakov A. V. 《Russian Journal of Plant Physiology》2001,48(3):273-280
All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H+-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H+-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H+-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H+-ATPase pumping in the plasmalemma through its influence on the coupling between H+-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process. 相似文献
17.
Suping Jiang Steven A. Anderson G. Douglas Winget Antony J. Mukkada 《Journal of cellular physiology》1994,159(1):60-66
Leishmania donovani has an active K+/H+ exchange system on the surface membrane. Modulation of external K+ concentration resulted in a corresponding change in internal pH (pHi) suggesting a link between proton and potassium transport. Although a Na+/H+ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K+/H+ exchange. Reduction of cellular ATP with NaN3 and KCN inhibits K+/H+ exchange showing thereby that the process is energy dependent. The K+/H+ exchange is sensitive to inhibitors of the gastric K+/H+-ATPase. It is concluded that the H+-ATPase previously reported on the plasma membrane of L. donovani is in fact a K+/H+-ATPase. © 1994 wiley-Liss, Inc. 相似文献
18.
Hai-Jun Gong Kun-Ming Chen Guo-Cang Chen Suo-Min Wang Cheng-Lie Zhang 《Plant Growth Regulation》2003,40(2):139-145
The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H+-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The Km for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H+-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H+-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H+-ATPase, therefore increasing the catalytic activity of the phosphatase domain. 相似文献
19.
Ana Maria Landeira-Fernandez Maricilia S. Costa Leopolde de Meis 《Bioscience reports》1996,16(6):439-451
Vesicles derived from maize roots retain a membrane bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H+-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent Km for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mol Pi/mg · 30 min–1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mol Pi/mg · 30 min–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+>Na+Li+).Abbreviations Mops
4-morpholinopropanesulfonic acid
- EDTA
ethylenediaminetetraacetic acid
- ACMA
9-amino-6-chloro-2-methoxyacridine
- FCCP
carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone 相似文献
20.
Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue
Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pK
a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes V
max but not K
m values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM. 相似文献