壳梭孢素对质膜H^＋—ATPase活力影响机制的研究进展

壳梭孢素（FC）作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统,FC刺激质膜H^ -ATPase的活性是通过FC结合蛋白（FCBP）与H^ -ATPase发生作用,FCBP是14－3－3蛋白家族成员之一。     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H⁺-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H⁺-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H⁺-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H⁺-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H⁺-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H⁺-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H⁺-ATPase. Analysis of the activation state of PM H⁺-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.     

N-Ethylmaleimide Modifies the Conformation of the Plasma Membrane H+-ATPase,Strengthening the Inhibitory Action of the C-terminal Domain1

Abstract: The effect of cysteine modification with N-ethylmalei-mide (NEM) on the activity of the plasma membrane (PM) H+-ATPase and on its activation state was investigated in PM isolated from aged red beet parenchyma slices. Treatment of PM with increasing concentrations of NEM (0.1–1mM) drastically reduced H+-ATPase activity. The inhibiting effect of PM treatment with NEM was stronger when the H+-ATPase activity was assayed at pH values (7.1–7.2) higher than that optimal for enzyme activity (6.3). If the PM H+-ATPase was activated by proteolytic cleavage of the C-terminal domain or by its displacement by fusicoccin prior to NEM treatment, the inhibitory effect of NEM on the W-ATPase activity became independent of the pH of the assay medium. Moreover, inhibition by NEM of H+-ATPase activity also became independent of the pH of the assay medium if the C-terminal was proteolytically cleaved or displaced by lysophosphatidylcholine after NEM treatment of the PM. Controlled trypsin treatment of NEM-treated PM produced, beside the 90 kDa truncated PM H+-ATPase, fragments of 60 to 30 kDa of the enzyme that were undetectable after trypsin treatment of control PM. These results indicate that PM treatment with NEM modifies the H+-ATPase conformation, exposing trypsin cleavage sites scarcely accessible in control PM and strengthening the autoinhibitory action of the C-terminal domain.     

壳梭孢素对质膜H+-ATPase活力影响机制的研究进展

壳梭孢素（FC）作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统,FC刺激质膜H+_ATPase的活性是通过FC结合蛋白（FCBP）与H+_ATPase 发生作用。FCBP是14-3-3蛋白家族成员之一。     

壳梭孢素对质膜H~ -ATPase活力影响机制的研究进展

壳梭孢素 (FC)作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统 ,FC刺激质膜H _ATPase的活性是通过FC结合蛋白 (FCBP)与H _ATPase发生作用。FCBP是 1 4_3_3蛋白家族成员之一     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

植物质膜H+-ATPase响应盐胁迫的分子机制

植物细胞质膜质子泵（PM H^＋-ATPase）有看家酶之称,是由多基因编码的,其主要功能是向细胞营养物质的吸收和离子跨膜运输提供驱动力。文章介绍PM H^＋-ATPase在植物抗盐中的作用及研究进展。     

植物细胞质膜H+-ATPase的结构与功能

植物细胞质膜H+-ATPase属于P型质子泵。由该酶产生的跨膜电化学梯度是物质跨膜运输的原初动力。研究表明,质膜H+-ATPase与植物的生长发育密切相关,被称为植物细胞的“主宰酶”。近年,关于该酶的生化特性,基因表达与调控以及结构与功能等方面的研究取得重要进展。对质膜H+-ATPase的生化特性,分子结构,调节机制和生理功能等进行了综述。     

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H⁺-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H⁺-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H⁺-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.     

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

The effect of controlled proteolysis on the plasma membrane (PM)Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125l-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase co-migrates with the phosphorylated intermediate of the enzyme[mdash]labeled by incubation with [[gamma]-32P]GTP in the presence of calcium[mdash]on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme.     

Modulation of H+-ATPase Activity by Fusicoccin in Plasma Membrane Vesicles from Oat (Avena sativa L.) Roots (A Comparison of Modulation by Fusicoccin,Trypsin, and Lysophosphatidylcholine)

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM vesicles isolated from oat (Avena sativa cv Adamo) roots by aqueous two-phase partitioning. Considerable stimulation of activity was observed only when roots were treated with fusicoccin prior to the PM isolation. Fusicoccin treatment shifted the pH optimum of the ATPase toward more alkaline values and increased Vmax. No effects on Km were observed. Treatment with trypsin resulted in stimulation of ATPase activity in control vesicles but not in the fusicoccin-treated vesicles. The characteristics of stimulation by trypsin in control vesicles were comparable with those of stimulation by fusicoccin. This result and the change of the polypeptide pattern on western blots suggest the involvement of the C-terminal inhibitory domain in the fusicoccin signal transduction chain. On the other hand, stimulation by lyso-PC demonstrated other characteristics than stimulation by fusicoccin. Lyso-PC was able to stimulate ATPase activity at both acidic and alkaline pH values. Kinetic analysis of the pH dependency curves revealed different mechanisms for activation by fusicoccin and by lyso-PC. Whereas fusicoccin shifted the pH dependency of formation of phosphorylated intermediate to more alkaline values, lyso-PC seemed to increase dephosphorylation independently of pH.     

Characterization of the Red Beet Plasma Membrane H+-ATPase Reconstituted in a Planar Bilayer System

The transport activity of the red beet (Beta vulgaris L.) plasma membrane H+-ATPase was examined following reconstitution into a planar bilayer membrane. Fusion of partially purified plasma membrane H+-ATPase with the bilayer membrane was accomplished by perfusion of proteoliposomes against the bilayer under hypoosmotic conditions. Following incorporation into the bilayer, an ATP-dependent current was measured that demonstrated properties consistent with those of the plasma membrane H+-ATPase. Current production was substrate specific for ATP, inhibited by orthovanadate, and insensitive to 200 nM erythrosin B but inhibited by 100 [mu]M erythrosin B. When current production was measured as a function of Mg:ATP concentration, a simple Michaelis-Menten relationship was observed and a Km of 0.62 mM was estimated. Current-voltage analysis of ATP-dependent current in the presence of 0.5 mM ATP, 20 mM ADP, 40 mM orthophosphate, and an opposing 2.5-unit [delta]pH revealed a reversal potential of about -149 mV. Based on the free energy available from ATP hydrolysis, this reversal potential is consistent with an H+/ATP stoichiometry of 1. This study demonstrates the usefulness of a planar bilayer system for investigation of energy coupling to H+ transport by the plasma membrane H+-ATPase.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

Purification and Properties of a Plasma Membrane H+-ATPase from the Extremely Acidophilic Alga Dunaliella acidophila

This paper describes partial purification and characterization of a vanadate-sensitive H+-ATPase from plasma membranes of Dunaliella acidophila, an extremely acidophilic unicellular alga (I. Sekler, H.U. Glaser, U. Pick [1991] J Membr Biol 121: 51-57). Purification is based on the insolubility in and stability of the enzyme in Triton X-100. The purified enzyme is highly enriched in a polypeptide of molecular mass 100 kD, which cross-reacts with antibodies against the plant plasma membrane H+-ATPase. Upon reconstitution into proteoliposomes, the enzyme catalyzes an ATP-dependent electrogenic H+ uptake. ATP hydrolysis is stimulated by lipids, is inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide, erythrosine, and mercurials, and shows a sharp optimum at pH 6. Unusual properties of this enzyme, by comparison with plant plasma membrane H+-ATPases, are a higher affinity for ATP (Km = 40 [mu]M) and a larger stimulation by K+, which interacts with the enzyme from its cytoplasmic side. Comparative studies with cross-reacting antibodies, prepared against different domains of the plant H+-ATPase, suggest that the central hydrophilic domain containing the catalytic site is more conserved than the C- and N-terminal ends. The high abundance and stability of the plasma membrane H+-ATPase from D. acidophila make it an attractive model system for studies of the structure-function relations and regulation of this crucial enzyme.     

Characterization and Effect of Light on the Plasma Membrane H+-ATPase of Bean Leaves

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the K_m was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, K_m, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.