Characterization of a red beet protein homologous to the essential 36-kilodalton subunit of the yeast V-type ATPase.

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (Vo) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the Vo sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated Vo subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggest that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPases upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.

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.

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.

Reversal of the red beet tonoplast H(+)-ATPase by a pyrophosphate-generated proton electrochemical gradient.

The reversal of the tonoplast H(+)-ATPase to mediate ATP synthesis was investigated in tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue. Our approach involved use of the H(+)-PP(i)ase to establish a proton electrochemical gradient (delta muH+) across the tonoplast vesicle membrane to drive the H(+)-ATPase in reverse. However, an initial problem with this approach was the presence of an adenylate kinase activity in the tonoplast fraction that interfered with measurement of ATP synthesis as a coupling between the H(+)-ATPase and H(+)-PP(i)ase. Inclusion of the adenylate kinase inhibitor p1p5-di(adenosine)pentaphosphate (Ap5A) in assays at 50 microM led to a complete inhibition of this activity and allowed measurement of ATP synthesis coupled to PPi hydrolysis. When measured in the presence of Ap5A, PPi-dependent ATP synthesis was blocked by Triton X-100 and inhibited by gramicidin D, imidodiphosphate, nitrate, and bafilomycin A. These results are consistent with PPi-dependent ATP synthesis occurring as a coupled process involving a delta muH+ established across the membrane. Furthermore, the observation that ATP synthesis is inhibited by inhibitors of the tonoplast H(+)-ATPase (nitrate and bafilomycin A) would suggest that this enzyme is involved in the synthetic reaction and can operate in reverse to synthesize ATP from ADP and Pi. A thermodynamic analysis of coupling between the H(+)-PP(i)ase and H(+)-ATPase suggests that PPi-driven ATP synthesis could only occur under these reaction conditions if the H+/substrate stoichiometries for the H(+)-PP(i)ase and H(+)-ATPase were 1 and 2, respectively. These values are consistent with transport stoichiometries previously determined for these enzymes in red beet tonoplast vesicles using kinetic methods.

Energy transduction in tonoplast vesicles from red beet (Beta vulgaris L.) storage tissue: H+/substrate stoichiometries for the H(+)-ATPase and H(+)-PPase.

The H+/substrate stoichiometries of the tonoplast H(+)-ATPase and H(+)-PPase were determined by a kinetic approach. Using red beet (Beta vulgaris L.) tonoplast vesicles, rates of substrate-dependent H+ transport were estimated by (I) a mathematical model describing the time course of delta pH formation, (II) the rate of H+ leakage following H+ pump inhibition at a steady state delta pH, and (III) the initial rate of alkalinization of the external medium. When compared with rates of substrate hydrolysis measured under identical conditions, all three methods yielded an H+/ATP stoichiometry of 2 while the H+/PPi stoichiometry was determined to be 1 using methods I and II. Experimental limitations did not permit an analysis of the H+/PPi stoichiometry by method III. From these results and the estimated level of substrate and product typically found in the cytoplasm of plant cells, it is suggested that the H(+)-ATPase and H(+)-PPase as primary H(+)-pumps are poised toward net substrate hydrolysis under in vivo conditions thereby operating in parallel to generate a proton electrochemical gradient across the tonoplast.

Characterization of a H+/NO3- symport associated with plasma membrane vesicles of maize roots using 36ClO3- as a radiotracer analog.

The mechanism of NO3- transport was examined in isolated plasma membrane vesicles from maize (Zea mays L., hybrid B73 X LH 51) roots using 36ClO3- as a radiotracer analog for NO3-. When an acid-exterior delta pH was imposed across the vesicle membrane, uptake of 36ClO3- was stimulated and the time course of radiolabel uptake displayed an overshoot phenomenon characteristic of the coupling of one solute gradient ot the movement of another solute. Evidence supporting delta pH as the driving force for 36ClO3- uptake included a dependence of the overshoot peak and initial rate of 36ClO3- uptake on the magnitude of the imposed delta pH, the occurrence of delta pH-driven 36ClO3- uptake in the presence of KSCN/valinomycin, and the ability of an imposed delta pH to drive 36ClO3- uptake when radiolabel was equilibrated across the membrane. When delta pH-driven 36ClO3- transport was examined in the presence of NO3-, radiolabel uptake was inhibited in a competitive manner. This was consistent with the carrier having the capacity to use either ClO3- or NO3- and supports the use of this radiotracer as an analog for NO3- in transport studies. When delta pH-driven 36ClO3- uptake was examined as a function of 36ClO3- concentration and delta pH, saturation kinetics were observed and the magnitude of the imposed delta pH affected the Km but not the Vmax for 36ClO3- uptake. This suggested an ordered binding mechanism where 36ClO3- would bind to the protonated form of the carrier prior to translocation. Radiolabeled 36ClO3- uptake was inhibited by treatment of the vesicles with phenylglyoxal, suggesting the involvement of arginine moieties in the process of transport. Taken together, these results support the presence of a H+/NO3- symport carrier at the plasma membrane which could be involved in mediating energy-dependent NO3- uptake into plant cells.

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 (DeltaG(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.

Ca-translocating ATPase of the plant plasma membrane.

For Ca(2+) to function as a second messenger in signal transduction, it is essential that plant cells maintain low cytoplasmic Ca(2+) levels relative to internal organelles and the apoplast. At the plasma membrane, Ca(2+) is actively transported out of the cytoplasm and current evidence supports the involvement of a primary Ca(2+)-translocating ATPase in mediating this energy-dependent process. This review examines the preliminary biochemical characterization of this transport enzyme.

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.

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress.

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H(+)-transport activity were measured by quinacrine fluorescence (DeltapH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (DeltaPsi) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K(d)) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Psi(o)) of-26.0 and -13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K(d) values and the calculated intrinsic affinity constant, K(i). The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Psi(o) values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K(+)-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl(-) ions to stimulate H(+)-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate.

The GTP-driven component of Ca(2+) uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca(2+)-translocating ATPase and assess its utility as a probe for this transport system. Uptake of (45)Ca(2+) in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca(2+) concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca(2+)-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of (45)Ca(2+)-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven (45)Ca(2+) uptake represents the capacity of the plasma membrane Ca(2+)-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [gamma-(32)P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca(2+)-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca(2+)-ATPases present in animal cells.

A modified method for the production of 36ClO3- for use in plant nitrate transport studies.

A modified method for the production and purification of 36ClO3- in high yield is described. This procedure, involving the electrolytic oxidation of 36Cl- in a cell with simple electrode design and purification of the electrolysis products (36Cl-, 36ClO3-, and 36ClO4-) by aqueous column chromatography, allows for the recovery of about 80% of the initial radiolabel as 36ClO3-. The method is rapid and suitable for the production of this radiolabeled anion for use as a tracer analog for nitrate in plant membrane transport experiments.

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution.

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of gamma-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.

Modification of an essential arginine residue associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue.

The dicarbonyl compounds, phenylgloxyl and 2,3-butanedione were used to demonstrate the presence of an essential arginine residue in the mechanism of the red beet (Beta vulgaris L.) plasma membrane ATPase. Treatment of the red beet ATPase with either of these reagents resulted in an inhibition of ATP hydrolytic activity protectable by the inclusion of either ATP or ADP during inhibitor incubation. Ligands of the ATP hydrolytic reaction also protected against phenylglyoxyl inhibition and affected the ability of ADP to protect against inhibition by this reagent. Kinetic analysis of 2,3-butanedione and phenylglyoxyl inhibition suggested the presence of a single arginine residue susceptible to attack by these reagents. As similar results with these arginine modification reagents were found for both the plasma membrane-associated and solubilized forms of the ATPase, it is apparent that the function of this arginyl moiety is not affected by detergent treatment and removal of the enzyme from the membrane.

A small scale procedure for the isolation of transport competent vesicles from plant tissues.

A microscale method for the isolation of selectively sealed microsomal membrane fractions from plant tissue is presented. The method is based on differential centrifugation in a table top microcentrifuge to accommodate small sample size (10-25 g tissue) and the addition of KI or KCl in the homogenization medium for isolating selectively sealed plasma membrane or tonoplast vesicles. This microscale procedure was found to be useful in isolating membranes from red beet (Beta vulgaris) storage tissue, sugar beet (Beta vulgaris) storage tissue, corn (Zea mays) roots, and soybean (Glycine max) roots. This paper also describes the ability to further purify an enriched red beet plasma membrane fraction on a discontinuous sucrose density gradient, in a microcentrifuge, that is highly competent in ATP-dependent H+-transport. The speed and wide applicability of this procedure make it ideal when a large number of samples need to be processed.

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea: II. Partial characterization of Ca2+ transport and glyceollin effects.

Calcium uptake was examined in sealed plasma membrane vesicles isolated from the plant pathogenic fungus, Phytophthora megasperma f. sp. glycinea. Calcium uptake was ATP-dependent and by the addition of various ionophores in the presence of ATP, it was shown that Ca2+ transport was mediated by a nH+/Ca2+ antiport. Further evidence for this antiport mechanism included Ca2+ uptake driven by an imposed pH gradient and the observation that calcium could dissipate a steady-state pH gradient across the vesicle membrane. Transport mediated by the nH+/Ca2+ antiport was optimal at pH 7.0, and demonstrated saturation kinetics for Ca2+ with a Km of about 7 microM. Glyceollin, a soybean phytoalexin, was found to inhibit Ca2+ transport consistent with its ability to increase H+ conductance. In the presence of glyceollin, calcium leakage from Phytophthora membrane vesicles also increased. This study provides basic information about calcium transport in a plant pathogenic fungus as well as demonstrating a possible mode of action of a phytoalexin.

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity.

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.

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.

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state.

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([gamma-(32)P]ATP and [5',8-(3)H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg(2+)-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg(2+) increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.