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.     

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.     

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.     

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl⁻ and inhibited by NO₃⁻. NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K₂SO₄ supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K⁺. KCl and K₂SO₄ support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.     

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.     

Kinetics Analysis of the Plasma Membrane Sucrose-H+ Symporter from Sugar Beet (Beta vulgaris L.) Leaves

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Suc uptake increased 18-fold as the pHo increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Suc uptake remained constant. The effects of pHi in the presence or absence of internal Suc were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]i) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Suc transport might be sensitive to changes in pHo. On the other hand, the [H+]i for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Suc transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Suc on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).     

Chemical Equivalence of Phosphoenzyme Reaction States in the Catalytic Mechanism of the Red Beet (Beta vulgaris L.) Plasma Membrane ATPase

A comparison of two phosphoryl enzyme reaction states associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue was carried out to determine if their differences in reactivity toward ADP and K⁺ was related to a structural difference in the site of phosphorylation. Using a pulse labeling method it was possible to produce preparations where either the ADP-sensitive and -insensitive phosphoenzyme forms or the ADP-insensitive phosphoenzyme form alone were trapped as trichloroacetic acid denatured protein. Following complete digestion with Pronase, both preparations yielded radioactive tripeptides with similar properties with respect to pH stability of the covalent bond linking the phosphate to the peptide, isoelectric point, and migration on cellulose thin layer plates. Since the preparation containing both intermediate reaction states behaved in a uniform manner during analysis and displayed properties similar to the preparation containing only the ADP-insensitive phosphoenzyme form, it was proposed that both phosphoenzyme forms were chemically equivalent and derived from the same region of the catalytic active site. The observation that ethyleneimine treatment of both preparations followed by trypsin digestion resulted in the production of tripeptides similar to the Pronase fragments would support this proposal since it suggests that the tripeptides from both phosphoenyzme states contain a lysine residue on the C terminal end and are adjacent to a cysteine residue on the N-terminal end. The chemical equivalence of these two phosphoenzyme reaction states suggests that their differences in reactivity toward ligands may be related to conformational changes associated with the catalytic and transport mechanism of this enzyme.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.     

Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : A Comparative Study of Ion Effects on DeltapH and DeltaPsi

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon ΔpH and ΔΨ revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce ΔΨ. For tonoplast vesicles, the relative order for this effect was KI > KBr ≥ KCl > KClO₃ > K₂SO₄ while for plasma membrane vesicles, a different order KI > KNO₃ ≥ KBr ≥ KClO₃ > KCl > K₂SO₄ was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K⁺/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.     

Agrobacterium tumefaciens-Mediated Genetic Transformation of Sugar Beet (Beta vulgaris L.)

Genetic transformation of sugar beet (Beta vulgaris L.) cv Bella (2n = 3x = 27) cv Dipple Ero (2n = 2x = 18) and accession SVP31-188 (2n = 2x = 18) was investigated by Infecting in vitro-derived shoot base segments with Agrobacterium tumefaciens strain LBA 4404 carrying either pBin 19 or pSI 121 plasmid. MS media supplemented with 0.5 mg.l^-1 naphthalene acetic acid, 0.25 mg.l^-1 6-benzylaminopurine and 100 βg.ml^-l kanamycin were effective for the production of callus and shoots from Agrobacterium Infected explants irrespective of the method of infection. Most of pSin 19- and all of pSI 121-medlated transformants failed to root on selective rooting media. Only 11.1% of pSin 19-mediated Sella transformants rooted on media containing 0.5 mg.l^-1 kinetin and 100 βg.ml^-1 kanamycin and all of them were the product of co-cultivation In the presence of acetosyringone. In the case of pSI 121-mediated transformants as many as 40–50 copies of the GUS gene was determined to be integrated with tandem repeats into at least six different sites of cv Sella genome. Fluorometrlc assay also demonstrated the transformed nature of a number of sugar beet shoots.     

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.     

甜菜胞质型谷氨酰胺合成酶基因组DNA的克隆

以甜菜叶片为材料,用CTAB法提取基因组DNA.以分段PCR法扩增得到了完整的甜菜胞质型谷氨酰胺合成酶(GS1)基因组DNA.采用RT-PCR法扩增此GS1基因(GS1)的cDNA序列应用于对照.获得了长度为9 606bp的完整的GS1 DNA序列和长度为1 068 bp的GSI cDNA序列.分析GS1基因组DNA序列表明,它包含13个外显子,被12个内含子分隔开.其外显子区与已公布的GS1 mRNA序列的相似性达99.5%.RT-PCR法获得的cDNA序列与已知的GS1 mRNA序列相似性达99.6%.而2次实验中GS1基因组DNA外显子区与GS1 cDNA序列的相似性达99.9%.GenBank登录号为EU370974.     

Sucrose Phosphate Is Not Transported into Vacuoles or Tonoplast Vesicles from Red Beet (Beta vulgaris) Hypocotyl

Tonoplast vesicles and vacuoles isolated from red beet (Beta vulgaris L.) hypocotyl accumulated externally supplied [¹⁴C]sucrose but not [¹⁴C]sucrose phosphate despite the occurrence of sucrose phosphate phosphohydrolytic activity in the vacuole. The activities of sucrose synthase and sucrose phosphate synthase in whole cell extracts were 960 and 30 nanomoles per milligram protein per minute, respectively; whereas, no sucrose synthesizing activity was measured in tonoplast preparations. The results obtained in this investigation are incompatible with the involvement of sucrose phosphate synthase in the process of sucrose synthesis and accumulation in the storage cells of red beet.     

Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots

In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met₃SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met₃SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met₃SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met₃SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met₃SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

甜菜NADH-NR基因的克隆和序列分析

利用同源序列克隆方法从标准偏高糖型甜菜品种甜研7号(Ty7)中获得氯素诱导NADH-NR基因片段,通过RACE技术克隆NADH-NR基因全长序列.该基因ORF长度2 718 bp,编码905个氨基酸,包括147 bp的5'UTR和382 bp的3' UTR,GenBank上的注册号为EU163265,基因编码蛋白的等电点为6.12,推测分子量大小为102 kD,C端(778-891 aa)有一个跨膜区域.基因编码多肽含有3个氧化还原功能区:钼辅因子功能区(eukary NR Moco,93 - 478aa),Fe-血红素结合区(Cytb5,535 -608 aa),FAD结合区(FAD binding 6,653 -760 aa).在甜研7号NR下游的氨基酸残基中含有NADH-NR特有的CGPPP-M基序,说明该蛋白以NADH为电子供体.通过比对,甜研7号的NADH-NR基因与菠菜NADH-NR基因同源性最高,为86.18％.经Southem杂变检验,甜研7号中NADH-NR基因以低拷贝数存在.经基因组克隆分析,甜研7号NADH-NR含有3个内含子,4个外显子.     

Characteristics of Sucrose Transport and Sucrose-Induced H+ Transport on the Tonoplast of Red Beet (Beta vulgaris L.) Storage Tissue

Sucrose-induced changes of the energization state of the red beet root (Beta vulgaris L. ssp. conditiva) vacuolar membrane were observed with the fluorescent dyes 6-chloro-9-{[4-(diethylamino)- 1-methylbutyl]-amino}-2-methoxyacridine dihydrochloride, as a pH monitor, and 9-amino-6-chloro-2-methoxyacridine (ACMA). Consequently, transient acidification of the surrounding suspension medium could be measured with a pH electrode. This signal was specific for Suc and was not seen for sorbitol, mannitol, or maltose. Sucrose-induced medium acidification was sensitive to the same inhibitors that were efficient in inhibiting sucrose transport, including the monoclonal antibodies TNP56-12 and C50-5-3. It was seen with vacuoles and vesicles energized with MgATP before sucrose was added but also with vacuoles not artificially energized previously. Using bafilomycin A1 for the inhibition of the vacuolar ATPase of vacuoles previously energized by MgATP, apparent Km values for H+ export from the vacuoles to the medium could be calculated taking into account the passive proton leak. Apparent Km values for H+ export determined from data obtained with pH measurements in the medium and with ACMA corresponded to those obtained previously for sucrose uptake. Comparing sucrose uptake rates with corresponding H+ export rates at the respective sucrose concentrations and at Km, a stoichiometry of approximately one proton per transported sucrose was estimated.     

Purification of an Acid Invertase from Washed Discs of Storage Roots of Red Beet (Beta vulgaris L)

The purification of an acid invertase from washed discs of storageroots of red beet (Beta vulgaris L.) is described. An overallpurification of 1210-fold was obtained using a combination of(NH₄)₂SO₄ precipitation, size-exclusion chromatography, ion-exchangechromatography, conA-sepharose chromatography and two roundsof FPLC on Mono Q HR 5/5, the first at pH 7·5, the secondat pH 6·5. The purified enzyme had a specific activityof 206