A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

Gould, J.Michael; Winget, G.Douglas

doi:10.1016/0003-9861(73)90015-5

Cited by 32 publications

(5 citation statements)

References 32 publications

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“…3C), and it can utilize sugar phosphates, pNPP, 4-MUP, or PPi as a substrate. Although an alkaline inorganic pyrophosphatase activity is associated with spinach thylakoids (16), this differs from thylakoid alkaline phospha- Plant Physiol. Vol.…”

Section: Kinetic Properties Of Alkaline Phosphatasementioning

confidence: 94%

Isolation and Characterization of an Alkaline Phosphatase from Pea Thylakoids

Kieleczawa

Coughlan²,

Hínd³

1992

Plant Physiol.

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Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem 11 in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett 1 [1980] ). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone II-S and phosphorylated, photosystem Il-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.properties of the protein kinase and phosphatase involved in this regulatory mechanism have been studied in thylakoid preparations, but a better understanding of the system has awaited the resolution and in vitro characterization of its component enzymes. The protein kinase from spinach thylakoids has been purified and characterized (11). Although several acid phosphatases have been isolated from thylakoid preparations (28,30,33), their activity is predominantly toward soluble phosphate esters rather than phosphoproteins. Furthermore, the stroma ranges from about pH 7.0 to 8.0 in the dark and light, respectively (32), and LHC-II dephosphorylation in situ has an optimum pH of 8.0 (4, 23). Thus, catalysis of dephosphorylation by a phosphatase with an alkaline pH optimum may be presumed.An enrichment strategy based upon maximizing the ratio of alkaline to acid phosphatase activity in thylakoid subfractions led to the successful purification of an alkaline phosphatase whose isolation and preliminary enzymological properties are described below. MATERIALS AND METHODSThe efficient utilization of absorbed light quanta in steadystate photosynthesis involves a mechanism for sensing the relative excitation rates of the two photosystems and optimally adjusting their electron throughputs. First 2Abbreviations: LHC-II, light-harvesting Chl a/b protein complex of PSII; AS, ammonium sulfate; CHAPS, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonic acid; MEGA-9, N-nonanoyl-N-methylglucamine; 4-MUP, 4-methylumbelliferyl phosphate; pNPP, p-nitrophenyl phosphate; TN, Tricine-NaOH, pH 8.0. MaterialsPea plants (Pisum sativum L. cv Progress ...

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Section: Kinetic Properties Of Alkaline Phosphatasementioning

confidence: 94%

Isolation and Characterization of an Alkaline Phosphatase from Pea Thylakoids

Kieleczawa

Coughlan²,

Hínd³

1992

Plant Physiol.

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“…Although considered specific to vacuolar membranes, an inactive form of the PP i ase has been found in different types of endomembranes ( Oberbeck et al 1994) and an active form has now been identified on the plasmalemma of rhizomatous ( Petel and Gendraud 1989) or cotyledonary cells ( Macrì and Vianello 1990; Robinson et al 1996; Long et al 1997). A membrane‐bound alkaline inorganic PP i ase has also been isolated from chloroplasts ( Gould and Winget 1973), but the first and most extensively studied H + ‐PP i ase is that identified on the chromatophore membranes of Rhodospirillum rubrum , whose features are summarized in a recent review ( Baltscheffsky and Baltscheffsky 1992).…”

Section: Introductionmentioning

confidence: 99%

Proton pumping pyrophosphatase from higher plant mitochondria

Vianello

Macrı̀

1999

Physiologia Plantarum

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In higher plant cells, there are some enzymes capable of 35-kDa protein which is loosely bound to the inner membrane. This protein exhibits a PP i ase activity, stimulated by phospho-utilizing pyrophosphate (PP i ) as an energy donor. Among these, membrane-bound proton pumping pyrophosphatases lipids, with characteristics very similar to the membrane-(H + -PP i ase) have been identified. In addition to the well-bound enzyme. The mitochondrial PP i ase is distinct from the V-PP i ase, because an antibody raised against the 35-kDa known vacuolar H + -PP i ase (V-PP i ase), there is evidence for protein does not react with tonoplast membranes. The mito-the presence of a mitochondrial H + -PP i ase. This enzyme is chondrial H + -PP i ase seems to have an F-type structure, localized on the inner surface of the inner membrane and similar to the F-ATP synthase and the membrane-bound catalyzes the specific hydrolysis of PP i , coupled to proton transport, with a H + /PP i stoichiometry of ca 2. This activity PP i ases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PP i , this enzyme may act as a is Mg 2 + -requiring, is stimulated by monovalent cations, and is inhibited by Ca 2 + , F − and diphosphonates. The H + -buffer for the electrochemical proton gradient, by hydrolyzing PP i, during conditions of oxygen deprivation. PP i ase contains a catalytic head which is constituted by a (DmH + ) across the vacuolar membrane; sucrose degradation via sucrose synthase, UDP-glucose pyrophosphorylase and fructokinase; entry into glycolysis via the pyrophosphate:fructose-6-phosphate phosphotransferase (Taiz 1986, Stitt 1998; Fig. 1). These reactions are ubiquitous in higher plants and their activities are often high, albeit the precise significance and role for plant metabolism and growth is still enigmatic (Stitt 1998). This uncertainty arises in part from the observation that these PP i -consuming reactions, apart from operating in vivo near to equilibrium, may be replaced by corresponding ATP-utilizing enzymes. Therefore, it is difficult to understand whether they are acting in parallel with the ATP-dependent reactions or as PP i -generating enzymes.The characterized phosphoanhydride-energized H + pumps may be subdivided into three major categories: F, P, and V, on the basis of the mechanism of action, inhibitor sensitivity and subunit organization (Pedersen and Carafoli 1987). By contrast, membrane-bound PP i ases are a new category of proton pumps (Rea et al. 1992), recognized to be present in all types of higher plant cells that have been examined. The vacuolar H + -PP i ase (V-PP i ase) is the best characterized of these and this subject is covered by excellent reviews Sanders 1987, Rea and

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“…Membrane-bound pyrophosphatase activity in R. rubrum chromatophores was first shown in 1964 [9] and has since been found in a number of organisms [10][11][12][13][14]. A method developed recently [15] for continuous monitoring of inorganic pyrophosphate (PPi) facilitates the very close study of PPi synthesis.…”

Section: Introductionmentioning

confidence: 99%

Demonstration of ΔpH‐ and Δψ‐induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

Strid¹,

Karlsson²,

Baltscheffsky³

1987

FEBS Letters

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It is possible to obtain synthesis of PPi by artifical ion potentials in Rhodospirillum rubrum chromatophores. PPi can be formed by K +-diffusion gradients (A ~,), H + gradients (ApH) or a combination of both. In contrast, ATP can only be synthesized by imposed A~u or d~,+dpH. For ATP formation there is also a threshold value of K + concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP~ formation. Both PPi and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PPi can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PPi is 200 nmol PPi//tmol BChl, while that of ATP is no more than 25 nmol ATP/Itmol BChl. The initial rates of syntheses were 0.50/tmol PPd/lmol BChl per min and 2.0/tmol ATP/~tmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.Acid-base jump; Bioluminescence; H +-ATPase; H +-Pyrophosphatase; K +-diffusion potential; (Rhodospirillum rubrum)

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A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

Cited by 32 publications

References 32 publications

Isolation and Characterization of an Alkaline Phosphatase from Pea Thylakoids

Isolation and Characterization of an Alkaline Phosphatase from Pea Thylakoids

Proton pumping pyrophosphatase from higher plant mitochondria

Demonstration of ΔpH‐ and Δψ‐induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

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