The Plasma Membrane ATPase of Plant Cells: Cation or Proton Pump?

Leonard, Robert T.

doi:10.1007/978-1-4684-4085-0_91

Cited by 14 publications

(23 citation statements)

References 23 publications

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“…The observed stimulation of the ATP hydrolytic activity of the plasma membrane H+-ATPase by K+ has also been considered as evidence for a direct role in K+ uptake (Leonard, 1982;Briskin and Hanson, 1992, and refs. therein).…”

Section: Discussionmentioning

confidence: 99%

Role of the Plasma Membrane H+-ATPase in K+ Transport

Briskin

Gawienowski

1996

Plant Physiology

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The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Befa vulgaris 1.) 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 1 O0 PM vanadate or 1 O PM carbonyl cyanide-ptrifluoromethoxyphenylhydrazone. 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, protonophoreinsensitive component of ATP-dependent K+ transport i n 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.The plant plasma membrane H+-ATPase links ATP hydrolysis to the extrusion of protons from the cytoplasm to the cell exterior (Briskin, 1990;Michelet and Boutry, 1995). This provides a driving force for solute transport at the plasma membrane, consisting of an acid-exterior pH gradient and interior-negative electrical potential difference (Briskin and Hanson, 1992, and refs. therein). Furthermore, the plasma membrane H+-ATPase has a major role in a number of important physiological processes, including cell elongation (Rayle and Cleland, 1992), stomatal movements (Assmann, 1993), and cellular responses to a number of factors, including plant growth regulators, light, and funga1 toxins (Briskin, 1990; Palmgren, 1991, and refs. therein;Briskin and Hanson, 1992).A characteristic property of the plant plasma membrane H+-ATPase is the stimulation of its activity by K t (Briskin, 1990; Briskin and Hanson, 1992, and refs. therein). In early studies of the plasma membrane H+-ATPase, it was suggested that this K+ stimulation of activity might reflect the direct transport of this cation by the enzyme (Leonard,

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Section: Discussionmentioning

confidence: 99%

Role of the Plasma Membrane H+-ATPase in K+ Transport

Briskin

Gawienowski

1996

Plant Physiology

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“…It is widely believed that an ATPase associated with the plasma membrane ofhigher plant cells may be involved in driving energydependent proton efflux (9,10,12). While the plant plasma membrane ATPase has not been fully purified, its properties have been well characterized in partially purified membrane fractions from a limited number of species (4,9,10).…”

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confidence: 99%

“…While the plant plasma membrane ATPase has not been fully purified, its properties have been well characterized in partially purified membrane fractions from a limited number of species (4,9,10). Similar to the cation transport ATPases of animal cells (7) and the plasma membrane H+-ATPase of fungal cells (6), the plant ATPase forms a phosphorylated intermediate on a 100,000 D polypeptide when incubated with [y-32PJATP (2,3,5).…”

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Role of Magnesium in the Plasma Membrane ATPase of Red Beet

Briskin

Poole²

1983

Plant Physiol.

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The phosphorylation technique was used to assess the role of Mg in the red beet (Beta vulgans 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 exponentiaHly and the rate constant for dephosphorylation was similar to that observed when phosphorylation reactions were chased with unlabeled ATP. When KCI was included with the EDTA chase, a 2. It is widely believed that an ATPase associated with the plasma membrane ofhigher plant cells may be involved in driving energydependent proton efflux (9,10,12). While the plant plasma membrane ATPase has not been fully purified, its properties have been well characterized in partially purified membrane fractions from a limited number of species (4, 9, 10). Similar to the cation transport ATPases of animal cells (7) and the plasma membrane H+-ATPase of fungal cells (6), the plant ATPase forms a phosphorylated intermediate on a 100,000 D polypeptide when incubated with 3,5). This property is in contrast to the proton pumping ATPases ofprokaryotes and organelles which are not phosphorylated when ATP is hydrolyzed (1 1).The plasma membrane ATPase from higher plants is Mg activated and further stimulated by monovalent cations (9, 10). Whether or not this stimulation of the activity by monovalent cations is a manifestation of direct transport of these species by the enzyme is as yet unresolved (10). The requirement for Mg is believed to occur because the true substrate for the enzyme is the Mg-ATP complex (1). This role of Mg has been proposed for a number of the transport ATPases from animal cells (7) zyme has been proposed for the Ca2+-ATPase of the sarcoplasmic reticulum (7) and red cell membrane (14). In this report, the phosphorylation technique was used to clarify the specific role of Mg in activating the ATPase of red beet plasma membranes. MATERIALS AND METHODSRed beet (Beta vulgaris L.) storage roots were purchased commercially. The tops of the plants were removed and the storage roots were stored for up to 3 months in moist vermiculite at 5°C. Plasma membrane-enriched fractions were isolated as previously described (4) and immediately frozen under liquid N2 for up to 4 d without significant loss in ATPase activity. ATPase phosphorylation was measured at pH 6.5 and ice temperature by the incorporation of radioactive phosphate from [y-32P]ATP into TCA-insoluble protein as previously described (2, 3, 5). The details of the phosphorylation conditions are given for each experiment. The data shown represent the mean values for experiments which were repeated three times. RESULTS AND DISCUSSIONIn previous work, it was demonstrated that the plasma membrane ATPase from red beet required Mg ion and maximal activity occurred when Mg and ATP were present at the same concentration (4). Although these results from steady-state ATPase assays imply a substrate-related role for Mg (1), it is impossible to state the precise function of this cation i...

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“…Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO4 at pH 6.5 and then treated with sodium l3Hlborohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCI at 110°C for 22 hours There is strong evidence for the presence of an ATP-dependent, electrogenic proton efflux pump at the plasma membrane of higher plant cells (17 and references therein) and it has been postulated that a plasma membrane-associated ATP phosphohydrolase (ATPase) may represent the enzymic machinery responsible for this process (13,14). The plasma membrane ATPase isolated from corn roots (2-4), oat roots (18), and red beet storage tissue (6) has been shown to form a rapidly turning over phosphorylated intermediate on a 100,000 D polypeptide, presumably the catalytic subunit of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Evidence for a β-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

Briskin¹,

Poole²

1983

Plant Physiol.

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A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgani L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO4 at pH 6.5 and then treated with sodium l3Hlborohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCI at 110°C for 22 hours There is strong evidence for the presence of an ATP-dependent, electrogenic proton efflux pump at the plasma membrane of higher plant cells (17 and references therein) and it has been postulated that a plasma membrane-associated ATP phosphohydrolase (ATPase) may represent the enzymic machinery responsible for this process (13,14). The plasma membrane ATPase isolated from corn roots (2-4), oat roots (18), and red beet storage tissue (6) has been shown to form a rapidly turning over phosphorylated intermediate on a 100,000 D polypeptide, presumably the catalytic subunit of the enzyme. This property is similar to what has been observed for the animal cell Na+, K+-ATPase (12), sarcoplasmic reticulum Ca2+-ATPase (12), and the fungal cell plasma membrane H+-ATPase (11).The phosphorylated intermediate of the plasma membrane ATPase from corn roots (2), oat roots (18), and red beet storage tissue (6) is acid stable, alkali labile, and hydrolyzed by hydroxylamine. These properties suggest that the protein-phosphate bond is an acyl phosphate linkage formed by the side chain ofa glutamic or aspartic acid residue in the active site of the enzyme (20). The acyl phosphate bond is too labile to allow a direct determination of the phospho-amino acid in these intermediates (20) the acyl phosphate bond with sodium [3H]borohydride followed by protein digestion in acid and analysis of the labeled hydroxyamino acid. In this report, the borohydride reduction method was used to identify the catalytic phosphorylated amino acid residue in the plasma membrane ATPase from red beet storage tissue. MATERIALS AND METHODSPlant Material. Red beet (Beta vulgaris L.) storage roots were purchased commercially. The tops of the plants were removed and the storage roots were stored for un to 3 months in moist vermiculite at 5oC.Isolation of Plasma Membrane Fractions. Plasma membraneenriched fractions were isolated from red beet storage tissue as previously described (5) with minor modifications. The homogenizing medium was modified from that previously described (5) by the addition of 15 mms ,/-mercaptoethanol. The presence of ,Bmercaptoethanol tended to reduce some of the variability in the levels of ATPase activity observed previously (5). Plasma membrane fractions isolated from sucrose gradients were diluted with 250 mm sucrose, 1 mm Tris Mes (pH 7.2), 1 mm dithioerythritol (suspension buffer) to 28 ml, and then centrifuged at 227,000g (47,000 rpm) for 30 min in a Beckman Ti7O rotor. The resultant pellets were suspended in suspension buffer at a protein concentration of about 10 mg/ml and then frozen under liquid N2 for up to 4 d without ...

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The Plasma Membrane ATPase of Plant Cells: Cation or Proton Pump?

Cited by 14 publications

References 23 publications

Role of the Plasma Membrane H+-ATPase in K+ Transport

Role of the Plasma Membrane H+-ATPase in K+ Transport

Role of Magnesium in the Plasma Membrane ATPase of Red Beet

Evidence for a β-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

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