Immunocytolocalization of Plasma Membrane H-ATPase

The localization of plasma membrane H⁺-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a `two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H⁺ currents, which leave nongrowing parts and enter growing tips.     

Modification of the Red Beet Plasma Membrane H-ATPase by Diethylpyrocarbonate

Incubation of the red beet (Beta vulgaris L.) plasma membrane H⁺-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects.     

Inhibition and Labeling of the Plant Plasma Membrane H-ATPase with N-Ethylmaleimide

H⁺-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-³H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at M_r=100,000 and several other positions. However, only radioactivity in the M_r=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M_r=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H⁺-ATPase. When tryptic peptides are produced from N-[2-³H]ethylmaleimide labeled M_r=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-³H]ethylmaleimide incorporation is reduced in the presence of MgADP.     

Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H-ATPase from Red Beet Storage Tissue

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H⁺-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H⁺-ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca²⁺. Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H⁺-ATPase and that specific protein kinase(s) are probably associated with the enzyme.     

Immunofluorescent Localization of Plasma Membrane H-ATPase in Barley Roots and Effects of K Nutrition

The plasma membrane H⁺-ATPase (PM-H⁺-ATPase) of barley (Hordeum vulgare L. cv Klondike) roots was assayed by cross-reaction on western blots and cryosections with an antibody against the PM-H⁺-ATPase from corn roots. Under conditions of reduced K availability, which have previously been shown to increase K influx by greater than 25-fold, there were only minor changes detected in PM-H⁺-ATPase levels. Antibody labeling of cryosections showed the relative distribution of PM-H⁺-ATPase among cell types in root tips and mature roots. Epidermal cells, both protoderm and mature root epidermis, including root hairs, had high levels of antibody binding. In mature roots, the stelar tissue showing the highest antibody binding was the companion cells of the phloem, followed by pericycle, xylem parenchyma, and endodermis.     

胡杨质膜的纯化及其H＋-ATPase活性的研究

用Dextran T-500, PEG 3350两相分配法分离并纯化了悬浮培养的胡杨细胞质膜.不同聚合物浓度(5.5%、5.7%、5.9%、6.1%、6.3%、6.5%)和KCl浓度(0、5、10、15 mmol/L)对分离效果影响的研究结果表明, 采用聚合物浓度为5.9%和无盐存在的两相分配体系可获得纯度较高的胡杨细胞质膜.纯化的质膜H^＋-ATPase的活力提高8倍,且酶定向程度较高,这为进一步研究胡杨细胞质膜特性及获得高纯度H^＋-ATPase提供了良好基础.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: IV. N,N'-Dicyclohexylcarbodiimide Binding and Inhibition of the Plasma Membrane H-ATPase

The molecular weight and isoelectric point of the plasma membrane H⁺-ATPase from red beet storage tissue were determined using N,N′-dicyclohexylcarbodiimide (DCCD) and a H⁺-ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [¹⁴C]-DCCD at 0°C, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [¹⁴C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H⁺-ATPase. An antibody raised against the plasma membrane H⁺-ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H⁺-ATPase to be 6.5.     

Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid

The activation of H⁺-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H⁺-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H⁺-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.     

Phosphorylation by Inorganic Phosphate of the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H⁺-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions.     

Cu2+ Reduction by Tomato Root Plasma Membrane Vesicles

Reduction of Cu2+ by plasma membrane vesicles isolated from tomato (Lycopersicon esculentum Mill.) roots was investigated. Plants were grown in hydroponic culture with complete nutrition for 4 weeks or were deprived of Fe for the last 7 d. Plasma membrane vesicles were prepared by aqueous two-phase partitioning. Reduction of Cu, Fe, and ferricyanide by plasma membrane vesicles was measured. An increase in the activity of all three pyridine-nucleotide-dependent activities was noted in plasma membrane preparations from Fe-deficient, compared to Fe-sufficient, plants. Solubilization and chromatographic separation of two plasma membrane electron transport systems indicated that the Fe-chelate reductase was probably responsible for reduction of Cu. Assays used a variety of Cu chelates, and for each the Cu activity in the assay was determined by the program Geochem PC. The rate of reduction of Cu correlated with the level of Cu activity, and results support the idea that free Cu2+ and not Cu chelates may serve as the true substrate for reduction. Reduction was observed only in assays in which Cu activity was equivalent to Cu-enriched or Cu-toxic soils. These results suggest that reduction of Cu by tomato root may have little or no physiological relevance under conditions experienced by the root in the soil.     

Effects of Inorganic Phosphate on the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The effects of inorganic phosphate on the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) were studied. ATPase activity was inhibited weakly and noncompetitively by phosphate. This anion also relieved the inhibition caused by vanadate by displacing it from the enzyme. From this effect, a dissociation constant for phosphate of 25 millimolar and an extrapolated activity at infinite phosphate concentration of 84% of the activity without inhibitors were calculated. The partial inhibition by phosphate indicates the existence of a catalytically active enzyme-phosphate complex. In the presence of 24% dimethylsulfoxide, the inhibition of ATPase activity by phosphate is much greater than in its absence. This suggests that the active enzyme-phosphate complex could be converted into a covalent phosphoenzyme through a dehydration promoted by the low water activity of the medium. The inhibitory ability of phosphate in 24% dimethylsulfoxide was dependent on the presence of potassium. Potassium ions increased both the affinity for phosphate and the inhibition caused by an infinite phosphate concentration, suggesting that potassium stimulates both phosphate binding and phosphoenzyme formation.     

猪特异性脂肪细胞膜蛋白基因cDNA 5′端的分子克隆

猪特异性脂肪细胞膜蛋白是近年来才被报道的一种可能与前脂肪细胞生长、发育和脂肪沉积调控有关的蛋白质。采用PT-PCR和PACE技术,首次获得猪特异性脂肪细胞蛋白基因cDNA 5‘端的克隆和序列。     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation

The properties of the plasma membrane H⁺-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L., cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37°C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K⁺ in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This `latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit.     

Molecular Characterization of the Glycated Plasma Membrane Calcium Pump

We have previously demonstrated (Diabetes 39:707–711, 1990) that in vitro glycation of the red cell Ca²⁺ pump diminishes the Ca²⁺-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca²⁺ pump (Biochem. J. 293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca²⁺ pump, or to a partial inactivation of all the molecules. Glycation decreased the V _max for the ATPase activity leaving unaffected the apparent affinities for Ca²⁺, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V _max for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca²⁺-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca²⁺-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated. Received: 9 March 1999/Revised: 11 May 1999     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

番茄泛素蛋白基因LeEBF1和LeEBF2的克隆表达分析

通过RACE和RT-PCR方法从番茄中克隆了LeEBF1（EIN3binding F-box protein1）和LeEBF2（EIN3binding F-box protein2）的全长cDNA序列,两个基因LeEBF1、LeEBF2全长分别是2866和2891bp,对序列的分析表明,它们的开放阅读框分别是1911和1995bp,编码区编码637和665个氨基酸残基,在氨基端含保守的F-box区域和在羧基端有14个亮氨酸重复单位,通过BLAST软件和DNAMAN分析表明这两个基因的氨基酸序列与拟南芥EBF1和EBF2有58.6%相似,同时又与其他物种的EBF蛋白的F-box区域比较有24.4%到73.2%的相近。Northern杂交指出：LeEBF1与LeEBF2在野生型和Nr的幼叶中的表达量高于成熟叶;当在果实发育期,LeEBF1与LeEBF2在青果期的表达量相比其他时期要弱。初步结果表明,LeEBF1与LeEBF2可能在番茄的生长发育中起着重要的作用。     

番茄泛素蛋白基因LeEBF1和LeEBF2的克隆表达分析

通过RACE和RT-PCR方法从番茄中克隆了LeEBF1（EIN3 binding F-box protein 1）和LeEBF2（EIN3 binding F-box protein 2）的全长cDNA序列,两个基因LeEBF1、LeEBF2全长分别是2 866和2 891 bp,对序列的分析表明,它们的开放阅读框分别是1 911和1 995 bp,编码区编码637和665个氨基酸残基,在氨基端含保守的F-box区域和在羧基端有14个亮氨酸重复单位,通过BLAST软件和DNAMAN分析表明这两个基因的氨基酸序列与拟南芥EBF1和EBF2有58.6％相似,同时又与其他物种的EBF蛋白的F-box区域比较有24.4％到73.2％的相近。Northern杂交指出：LeEBF1与LeEBF2在野生型和Nr的幼叶中的表达量高于成熟叶;当在果实发育期,LeEBF1与LeEBF2在青果期的表达量相比其他时期要弱。初步结果表明,LeEBF1与LeEBF2可能在番茄的生长发育中起着重要的作用。     

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

The H⁺/ATP stoichiometry was determined for the plasma membrane H⁺-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H⁺/ATP stoichiometry utilized a kinetic approach where rates of H⁺ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H⁺ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H⁺ leakage from a steadystate pH gradient after stopping the H⁺-ATPase or utilizing a mathematical model which describes the net transport of H⁺ at any given point in time. When the rate of H⁺ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H⁺/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (ΔG_atp), this value for H⁺/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.