Rapid purification and reconstitution of a plant vacuolar ATPase using Triton X-114 fractionation: Subunit composition and substrate kinetics of the H+-ATPase from the tonoplast of Kalanchoë daigremontiana

doi:10.1016/0005-2736(92)90229-F

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 1106, Issue 1, 29 April 1992, Pages 117-125

https://doi.org/10.1016/0005-2736(92)90229-F Get rights and content

Abstract

A rapid procedure for the purification and reconstitution into proteoliposomes of the H⁺-translocating ATPase of plant vacuolar membranes in reported. It involves fractionation of the tonoplast with Triton X-114, resolubilization of the ATPase with octyl glucoside in the presence of a mixture of phosphatidyccholine, phosphatidylserine and cholesterol (27:53:20, by weight), and removal of the detergent by gel-giltration. Starting with partially purified vacuolar membranes, the procedure can be accomplished in about 2 hours. It has been applied to the H⁺-ATPase from the crassulacean plant Kalanchoë daigremontiana, from which it yields vesicles with a specific ATPase activity of about 3 μmol/min per mg protein. The purified enzyme contains polypeptides of apparent molecular mass 72, 57, 48, 42, 39, 33 and 16 kDa; these polypeptide also co-sediment on centrifugation of the solubilized ATPase through glycerol gradients. The 16·kDa subunit is labelled with [¹⁴C]dicyclohexylcarbodiimide. There is no evidence for a larger ATPase subunit in this preparation. The reconstituted ATPase proteoliposomes undergo ATP-dependent acidification, which can be measured by quenching of the fluorescence of 9-aminoacridine. The initial rate of fluorescence quenching is a measure of the rate of H⁺ translocation, and is directly proportional to the vesicle protein concentration, so the preparation is suitable for studying the kineticas of the tonoplast H⁺-ATPase. The dependence of the rate of fluorescence quenching on the concentration of MgATP is well fitted by the Michaelis equation, with a K_m value about 30 μM. ATP can be replace by dATP, ITP, GTP, UTP or CTP, and Mg²⁺ by Mn²⁺ or Ca²⁺; kinematic parameters for these substrates are reported. In contrast, hydrolysis of MgATP shows complex kinetics, suggestive either of negative cooperativity between nucleotide-binding sites, or of two non-interacting catalytic sites. Both the hydrolytic and H⁺-translocating activities of the proteoliposomes are inhibited by nitrate, though not in parallel, the latter activity being the more sensitive. Both activities are inhibited in parallel by bafilomycin A₁, which does not produce complete inhibition; the bafilomycin-insensitive component has complex ATPase kinetics similar to those of the uninhibited enzyme.

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Three-dimensional map of a plant V-ATPase based on electron microscopy
2002, Journal of Biological Chemistry
Citation Excerpt :
The K. daigremontiana V-ATPase samples used in our analysis by electron microscopy were characterized for purity and activity by SDS-PAGE and activity assays, respectively. Fig. 1A shows the typical polypeptide pattern of the V-ATPase after purification that corresponded to what has been reported previously (23, 33). During recent work the D-subunit (34 kDa) and two E-subunit isoforms (32 and 33 kDa) have been identified in the K. daigremontianaV-ATPase by matrix-assisted laser desorption ionization-mass spectroscopy (34).
V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V₀), and a soluble catalytic (V₁) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V₀ and V₁ were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V₀changed with respect to V₁, and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly.
Structure, function and regulation of the plant vacuolar H<sup>+</sup>-translocating ATPase
2000, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Km values ranging from 200 to 810 μM have been reported for different plant V-ATPases [34,62,65–69]. For the substrate hydrolysis activity of the K. daigremontiana V-ATPase two Km values (770 μM and 2 μM) have been found [70], indicating the presence of at least two different catalytic centers at the enzyme or cooperativity between nucleotide binding sites. By relating nitrate-sensitive V-ATPase activity to immunologically determined V-ATPase protein amount in tonoplast vesicles isolated from Me.
The plant V-ATPase is a primary-active proton pump present at various components of the endomembrane system. It is assembled by different protein subunits which are located in two major domains, the membrane-integral V_o-domain and the membrane peripheral V₁-domain. At the plant vacuole the V-ATPase is responsible for energization of transport of ions and metabolites, and thus the V-ATPase is important as a ‘house-keeping’ and as a stress response enzyme. It has been shown that transcript and protein amount of the V-ATPase are regulated depending on metabolic conditions indicating that the expression of V-ATPase subunit is highly regulated. Moreover, there is increasing evidence that modulation of the holoenzyme structure might influence V-ATPase activity.
V-ATPase- is a major component of the Golgi complex in the scaly green flagellate Scherffelia dubia
1999, Protist
Highly purified membranes isolated from the Golgi complex of the scaly green flagellate Scherffelia dubia (Chlorophyta) were subjected to Triton X-114 two-phase partitioning. Proteins in the detergent phase were analyzed by 2D gel electrophoresis and a major protein of 66 kD (p66) was N-terminally sequenced. The complete cDNA sequence of p66 was obtained by 3’ RACE-PCR and screening of a cDNA library of S. dubia with a PCR probe derived from the 3’ RACE. Sequence analysis of the cDNA clone identified p66 as subunit A of V-ATPase. Other major proteins in the isolated Golgi complex were immunoreactive to heterologous antibodies raised against subunit B or the holoenzyme of VATPase. A polyclonal (anti-p66) antibody raised against a recombinant, bacterially expressed p66 fusion protein recognized p66 in the isolated Golgi complex in western blots and localized the antigen by immunogold electron microscopy mostly to the scale reticulum but also to the Golgi stack within the Golgi complex. Concanamycin A-sensitive (but bafilomycin A₁-insensitive) ATPase activity was present in the isolated Golgi complex, and monensin at 0.5-1 μM reversibly inhibited flagellar regeneration and resulted in swelling of Golgi cisternae. It is concluded that a functional V-ATPase is a major protein of the Golgi complex in S. dubia and is presumably associated with sorting processes at the endocytotic/exocytotic boundary of the Golgi complex.
Increase in Bcl-2 level promoted by CD40 ligation correlates with inhibition of B cell apoptosis induced by vacuolar type H<sup>+</sup>-ATPase inhibitor
1998, Experimental Cell Research
We have previously demonstrated that cell death of WEHI-231 cells induced by specific inhibitors of vacuolar type H⁺-ATPase (V-ATPase) occurs through apoptosis. CD40 is involved in regulating activation, differentiation, and apoptosis of B cells. Here we show that the CD40 ligation rescues WEHI-231 cells from apoptotic cell death induced by a specific V-ATPase inhibitor, concanamycin A. CD40 signaling with anti-CD40 antibody resulted in the induction of Bcl-2 and Bcl-XL proteins in WEHI-231 cells. Constitutive expression of Bcl-2 but not Bcl-XL inhibited concanamycin A-induced apoptosis. These findings suggest that the expression of Bcl-2 mediated through CD40 signaling rescues the apoptotic cell death induced by blockade of V-ATPase. Interestingly, the acidification of intracellular acidic compartments was completely inhibited when WEHI-231 cells were cultured with concanamycin A, even in the presence of anti-CD40 antibody. In addition, apoptosis in WEHI-231 cells induced by concanamycin A was strongly suppressed when cultured with imidazole, a cell-permeable base, suggesting that apoptosis induced by concanamycin A is preceded by intraacidification.
The Physiology, Biochemistry and Molecular Biology of the Plant Vacuolar ATPase
1997, Advances in Botanical Research
This chapter discusses the physiology, biochemistry, and molecular biology of the plant vacuolar ATPase. The transmembrane movement of solute particles by direct consumption of energy, available from dissociation of covalent phosphoric acid–ester bonds, is limited to a few simple cations such as H⁺ and Ca²⁺ and possibly Na⁺ and Cl^–, with the H⁺- and Ca²⁺-transporting ATPases at the plasma membrane. H⁺–ATPases in general are located in different membrane systems. They are important for cellular metabolism in different ways. The V–ATPase can be visualized in the electron microscope by preparation of negatively stained membrane vesicles or of replicas of freeze fracture surfaces of vesicle membranes. By pumping protons into the vacuole, the V–ATPase removes H⁺ ions from the cytosol and thus, it is part of a cytoplasmic pH–stat mechanism. It also energizes the tonoplast by establishing an electrochemical proton gradient across this membrane. Both of these functions are basic physiological requirements in plant cell biology.
Functional reconstitution of the tonoplast proton-ATPase from higher plants
1997, International Review of Cytology
Tonoplast proton ATPase (V-ATPase) is the most widely spread H⁺ pump in plants. The electrochemical gradient generated by the H⁺ pump provides the driving force for the secondary transport of amino acids, ions, sugars, and several metabolites. The V-ATPase has an apparent functional mass of 400–600 kDa and comprises at least 9 or 10 different subunits, of which the catalytic 67–73 kDa, the neucleotide-binding 55–62 kDa, proteolipids 95–115 and 16–17 kDa, and 44–29 kDa required for activity and assembly are universal components. Reconstitution of the V-ATPase complex into liposome has been successful. Reconstitution is convenient to assess whether any set of subunits associated with the V-ATPase is sufficient to couple ATP hydrolysis to proton pumping. In this review, we describe practical approach of reconstitution of the V-ATPase from mung bean hypocotyls into asolectin liposomes.

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¹: Present address: Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK.

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Regular paperRapid purification and reconstitution of a plant vacuolar ATPase using Triton X-114 fractionation: Subunit composition and substrate kinetics of the H+-ATPase from the tonoplast of Kalanchoë daigremontiana

Abstract

Kidney Int.

Kidney Int.

J. Biol. Chem.

Trends Pharm Sci.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

FEBS Lett.

J. Plant Physiol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Regular paper
Rapid purification and reconstitution of a plant vacuolar ATPase using Triton X-114 fractionation: Subunit composition and substrate kinetics of the H⁺-ATPase from the tonoplast of Kalanchoë daigremontiana