Target molecular size of the red beet plasma membrane ATPase

Radiation inactivation of the red beet (Beta vulgaris L.) plasma membrane ATPase was carried out using γ-ray radiation from a ¹³⁷Cs source. Inactivation of vanadate-sensitive ATPase activity by γ-ray radiation followed an exponential decline with increasing total dose, indicating a single target size calculated to have a molecular weight of about 228,000. Since the catalytic subunit of the red beet plasma membrane ATPase has been demonstrated to have a molecular weight of about 100,000 by dodecyl-sulfate gel electrophoresis following ³²P-phosphorylation, it is suggested that the native enzyme may exist, at least, as a dimer of catalytic subunits.     

Intermediate reaction states of the red beet plasma membrane ATPase

The phosphorylation reaction for the plasma membrane ATPase of red beet (Beta vulgaris L.) was examined in order to further understand the mechanism of this enzyme. The level of steady-state phosphorylation had a pH optimum of about 6.0 while ATPase activity (32Pi production) measured under identical conditions had a pH optimum of 7.0. Phosphoenzyme decomposition was accelerated as both the pH and temperature were increased. The former effect may account for the observed difference between the pH optimum for phosphorylation and ATPase. Although the kinetics of K+ stimulation of ATP hydrolysis have been observed to be complex, the kinetics of K+ stimulation of phosphoenzyme turnover were observed to be simple Michaelis-Menten. An antagonism was observed between MgATP and K+ for the stimulation of phosphoenzyme turnover. Increased MgATP concentration reduced the degree of K+ stimulation of phosphoenzyme turnover and ATPase activity. These effects could be explained by the observation that two forms of phosphoenzyme occur during ATP hydrolysis. One form is discharged by ADP while the other form is ADP insensitive. Potassium stimulation of phosphoenzyme breakdown occurs primarily because of effects on the ADP-insensitive phosphoenzyme form. These results are consistent with a mechanism of ATP hydrolysis involving interconversions of conformational states.     

Role of magnesium in the plasma membrane ATPase of red beet

The phosphorylation technique was used to assess the role of Mg in the red beet (Beta vulgaris L.) plasma membrane ATPase. When an excess of ethylenediaminetetraacetate (Tris salt, pH 6.5) was added to phosphorylation reactions at steady-state, the phosphorylation level declined exponentially and the rate constant for dephosphorylation was similar to that observed when phosphorylation reactions were chased with unlabeled ATP. When KCl was included with the EDTA chase, a 2.4-fold increase in the turnover of the phosphoenzyme was observed. Thus, the formation of the phosphorylated intermediate but not its breakdown requires free Mg to be present. When an excess of unlabeled ATP containing MgSO₄ was added to plasma membranes incubated for 20 seconds with [γ-³²P]ATP in the absence of MgSO₄, a burst of phosphorylation was observed that declined exponentially. The rate constant for this decline was similar to that observed for phosphoenzyme turnover after initial labeling in the presence of MgSO₄. Extrapolation of this kinetic plot to zero time indicated that ATP binding can occur when MgSO₄ is absent. It is proposed that Mg has a specific role in the transphosphorylation reaction of the terminal phosphate group of ATP to the enzyme.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.     

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 ([γ-³²P]ATP and [5′,8-³H]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 Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ 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.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

Plasma membrane ATPase of red beet forms a phosphorylated intermediate

When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [γ-³²P] ATP, 40 micromolar MgSO₄ at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.     

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.     

Mechanistic investigation on the temperature dependence and inhibition of corn root plasma membrane ATPase

The kinetics of corn root plasma membrane-catalyzed Mg-ATP hydrolysis may be satisfactorily described by a simple Michaelis-Menten scheme. It was found that the Km of the process was relatively insensitive to changes in temperature. This property allowed us to conveniently estimate the activation energy of the enzyme turnover process as approximately 14 kcal mol-1 in the temperature range of 10 to 45 degrees C. The enzyme activity was inhibited by the presence of diethystilbestrol (DES), miconazole, vanadate, and dicyclohexylcarbodiimide (DCCD). The inhibition caused by DES and miconazole was strictly uncompetitive and inhibition by vanadate was noncompetitive. The inhibition by DCCD showed a substrate concentration dependence, i.e., competitive at high and uncompetitive at low concentrations of Mg-ATP. The 1/V vs [I] plots suggested that there were different but unique binding sites for DES, vanadate, and miconazole. However, the modification of the plasma membrane by DCCD exhibited interaction with multiple sites. Unlike yeast plasma membrane ATPase, the enzyme of corn root cells was not affected by the treatment with N-ethylmaleimide. Although the enzyme activity was regulated by ADP, a product of the reaction, the presence of inorganic phosphate showed no inhibition to the hydrolysis of Mg-ATP.     

Dual effect of ADP on the activity of the plasma membrane H+‐ATPase of red beet

The effect of ADP on the activity of the plasma membrane (PM) H⁺‐ATPase of red beet ( Beta vulgaris L.) parenchyma discs was evaluated by analyzing the effect of increasing concentrations of ADP on the kinetics of the reaction. When the PM H⁺‐ATPase activity was assayed at pH 6.3, ADP behaved as a simple competitive inhibitor. When the activity was assayed at pH 7.1, ADP not only increased the apparent K_m for MgATP but also decreased the V_max of the reaction. When the C‐terminal domain of the PM H⁺‐ATPase was cleaved by controlled trypsin treatment or displaced by addition of lysophosphatidylcholine, only the competitive component of inhibition by ADP of the activity assayed at pH 7.1 was evident. The results are discussed in relation to the physiological relevance of the activation of the PM H⁺‐ATPase by displacement of the autoinhibitory C‐terminal domain.     

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.     

Regulatory effects of adenosine diphosphate on the activity of the plasma membrane ATPase of corn roots

Plasma membrane enriched microsomal fraction was isolated from corn root cells by sucrose density centrifugation. The ATPase activity as measured by the release rate of inorganic phosphate, was decreased by the presence of modifiers which included diethylstilbestrol, vanadate, N,N'-dicyclohexylcarbodiimide, and miconazole. The presence of ADP also decreased the rate of ATP hydrolysis. Furthermore, a preincubation of the membrane with ADP significantly reduced the inhibitory effects of these membrane ATPase modifiers. Since the modes of interaction of these modifiers with the enzyme are different, the results suggest that the binding of ADP may stabilize the plasma membrane ATPase in a modifier insensitive state.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.     

Potassium-selective channel in the red beet vacuolar membrane

In higher plants the vacuolar K(+)-selective (VK) channel was identified solely in guard cells. This patch-clamp study describes a 40 pS homologue of the VK channel in Beta vulgaris taproot vacuoles. This voltage-independent channel is activated by submicromolar Ca(2+), and is ideally selective for K(+) over Cl(-) and Na(+).     

Steady-state kinetics of the plasma membrane H+-ATPase from red beet (Beta vulgaris): its inhibition by vanadate and inorganic phosphate

The intial velocity vs ATP concentration curves obtained with the plasma membrane H⁺-ATPase from red beet ( Beta vulgaris L.) did not follow classical Michaelis-Menten kinetics. A rate equation containing second-order terms in ATP concentration in both the numerator and the denominator was used to obtain a significantly better fit to the data. The observed deviations from Michaelis-Menten kinetics were more pronounced in the presence of potassium ions. The inhibition caused by inorganic phosphate was partial. i.e. the ATPase activity extrapolated at an infinite phosphate concentration was not zero. In contrast, the inhibition produced by orthovanadate was nearly total. The inhibitions caused by both phosphate and vanadate were uncompetitive with respect to ATP and enhanced by potassium ions and high concentrations of dimethyl sulfoxide. a solvent used to lower the water activity of the reaction medium. The ATP-dependent proton transport was stimulated by potassium ions and was inhibited by phosphate only at high ATP concentrations. A kinetic mechanism, in which the H⁺-ATPase can adopt two conformations during its catalytic cycle and can form a ternary enzyme-ATP-phosphate complex able to hydrolyze bound ATP. is proposed to explain those results.     

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

Summary In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H⁺-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu⁺ and Pma⁺ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5 end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.Publication n^o 2456 from the Biology Directorate of the Commission of European Communities     

Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells.

The lack of specific inhibitors of the plasma membrane Ca2+ pump (PMCA) has made vanadate (VO3-), a non-specific inhibitor, an invaluable tool in the study of PMCA function. However, three important properties of vanadate as an inhibitor of the PMCA in intact cells, namely its speed of action in different experimental conditions, the reversibility of its inhibitory effects at different doses, and its dose-response, had never been characterized, despite extensive use. We report here the speed, reversibility and dose-response of PMCA inhibition by vanadate in intact human red cells. Near maximal inhibitory concentrations (1mM) in the red cell suspension blocked almost instantly the uphill Ca2+ extrusion by the PMCA, regardless of the intracellular Ca2+ concentration, cation composition of the external media, membrane potential or volume-stability of the cell. PMCA inhibition by vanadate, at concentrations of 10mM and 1mM, was not reversed by washing, resuspending, and incubating the cells for up to 2h in vanadate-free media. Vanadate inhibited PMCA-mediated Ca2+ efflux in intact red cells with a K1/2 of approximately 3 microM, a value similar to that described for the Ca2+-ATPase in isolated red cell membranes.