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.     

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.     

Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

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.     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Purification and characterization of the plasma membrane ATPase of Neurospora crassa

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet (Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H+-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H+-ATPase.     

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.     

Blue light effects on stomata are mediated by the guard cell plasma membrane redox system distinct from the proton translocating ATPase

Abstract. The effects of blue light on stomata are critically analysed. Blue-light-induced increase in stomatal conductance is preceded by membrane hyperpolarization, proton efflux, potassium uptake and malate synthesis in guard cells. Hypothetically, a flavin containing plasma membrane redox system can pump protons out of guard cells on illumination with blue light. It is proposed that this electrogenic proton pump requires NAD(P)H but does not involve ATP/ATPase.     

In vitro membrane association of the F0 polypeptides of the Escherichia coli proton translocating ATPase.

The F0 polypeptides a, b, and c of the H+-translocating ATPase associated with membranes when synthesized in vitro. This association occurred when the membranes were present either cotranslationally or post-translationally. In addition, the F0 polypeptides associated with liposomes. The membrane association seemed to be an insertion process since there was protection of polypeptides a and c from proteolysis. The in vitro insertion of the F0 polypeptides a, b, and c was independent of the synthesis of each polypeptide and of the F1 polypeptides.     

Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.     

The plasma membrane proton pump ATPase: the significance of gene subfamilies

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a central role in transport across the plasma membrane. As a primary transporter, it mediates ATP-dependent H(+) extrusion to the extracellular space, thus creating pH and potential differences across the plasma membrane that activate a large set of secondary transporters. In several species, the H(+)-ATPase is encoded by a family of approximately 10 genes, classified into 5 gene subfamilies and we might ask what can this tell us about the concept, and the evolution, of gene families in plants. All the highly expressed H(+)-ATPase genes are classified into only two gene subfamilies, which diverged before the emergence of present plant species, raising the questions of the significance of the existence of these two well-conserved subfamilies and whether this is related to different kinetic or regulatory properties. Finally, what can we learn from experimental approaches that silence specific genes? In this review, we would like to discuss these questions in the light of recent data.     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

Purification and properties of the h-translocating ATPase from the plasma membrane of tomato roots

The proton-translocating, plasma membrane ATPase was purified from tomato roots. At the final stage of purification approximately 80% of the protein was found in a single band with an apparent molecular weight of 90 kilodaltons. Cross-linking studies indicated that the ATPase normally exists as a trimer of catalytic subunits. No evidence was found for any additional subunits. The pH optimum for ATP hydrolysis by the purified protein was 6.5. Activity was stimulated by K⁺, especially at low pH, and inhibited by vanadate, N,N′-dicyclohexylcarbodiimide, and diethylstilbestrol; nitrate was weakly inhibitory. Activity was stimulated by lysolecithin but inhibited by sonicated phospholipids. The inhibition by lipids could be prevented if octylglucoside was added with the lipids; the combination of octylglucoside and lipids actually stimulated activity. The purified protein could be reconstituted into liposomes and catalyzed ATP-dependent, vanadate-sensitive proton translocation.     

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.     

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(+).     

Proton translocating ATPase in lysosomal membrane ghosts. Evidence that alkaline Mg2+-ATPase acts as a proton pump

Membrane ghosts were prepared from purified lysosomes (tritosomes) of rat liver by hypo-osmotic treatment. Mg2+-ATP-driven acidification was observed in the membrane ghosts using acridine orange as a fluorescent probe of the transmembrane pH gradient (delta pH). Its properties were the same as those of intact lysosomes reported previously (Ohkuma, S., Moriyama, Y., & Takano, T. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762; Moriyama, Y., Takano, T., & Ohkuma, S. (1982) J. Biochem. 92, 1333-1336). The H+-pump was found to be electrogenic with use of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol as a fluorescent membrane potential probe. Alkaline Mg2+-ATPase activity was also identified on the membranes. It showed a pH maximum of pH 8.0-8.5, a Km value for ATP of 0.36 mM and a Vmax of 0.41 units/mg protein at 30 degrees C. Its activity was inhibited by dicyclohexylcarbodiimide, tri-n-butyltin, azide and ADP, but not by ouabain or vanadate. It differed from mitochondrial F1F0-ATPase in sensitivities to N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and oligomycin. Since this alkaline Mg2+-ATPase activity is very similar to the H+-pump activity in its requirement for divalent cations, substrate specificity and sensitivities to various chemicals, it may act as a proton translocase (H+-pump). Possible mechanisms of action of some chemicals, such as 4-acetamide-4'-isothiocyanatostilbene-2,2'-disulfonic acid, that inhibited the H+-pump but not the alkaline Mg2+-ATPase, are discussed.