Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.     

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.     

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps.

Vacuolar-type H(+)-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H(+)-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M(r) 64? omitted?000 membrane polypeptide that specifically catalyzes Mg(2+)-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H(+)-translocation is phylogenetically more deeply rooted than previously thought.     

Proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: purification, characterization, and reconstitution.

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4 mumol of PPi hydrolyzed min-1 mg-1 at pH 7.5 and 20 degrees C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56,000. The oligomeric structure of the H(+)-PPi synthase is discussed.     

H+ -PPases: a tightly membrane-bound family.

The earliest known H+-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H+-PPi synthase (proton-pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-PPi synthase and two algal vacuolar H+-PPases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-PPases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-PPases are reviewed and compared with H+-ATPases and soluble PPases.     

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps

Vacuolar-type H⁺-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H⁺-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M_r 64 000 membrane polypeptide that specifically catalyzes Mg²⁺-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H⁺-translocation is phylogenetically more deeply rooted than previously thought.     

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase

Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R-->A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R-->A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F- inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+-PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.     

Alternative photophosphorylation,inorganic pyrophosphate synthase and inorganic pyrophosphate

This minireview in memory of Daniel I. Arnon, pioneer in photosynthesis research, concerns properties of the first and still only known alternative photophosphorylation system, with respect to the primary phosphorylated end product formed. The alternative to adenosine triphosphate (ATP), inorganic pyrophosphate (PPi), was produced in light, in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, when no adenosine diphosphate (ADP) had been added to the reaction mixture (Baltscheffsky H et al. (1966) Science 153: 1120–1122). This production of PPi and its capability to drive energy requiring reactions depend on the activity of a membrane bound inorganic pyrophosphatase (PPase) (Baltscheffsky M et al. (1966) Brookhaven Symposia in Biology, No. 19, pp 246–253); (Baltscheffsky M (1967) Nature 216: 241–243), which pumps protons (Moyle J et al. (1972) FEBS Lett 23: 233–236). Both enzyme and substrate in the PPase (PPi synthase) are much less complex than in the case of the corresponding adenosine triphosphatase (ATPase, ATP synthase). Whereas an artificially induced proton gradient alone can drive the synthesis of PPi, both a proton gradient and a membrane potential are required for obtaining ATP. The photobacterial, integrally membrane bound PPi synthase shows immunological cross reaction with membrane bound PPases from plant vacuoles (Nore BF et al. (1991) Biochem Biophys Res Commun 181: 962–967). With antibodies against the purified PPi synthase clones of its gene have been obtained and are currently being sequenced. Further structural information about the PPi synthase may serve to elucidate also fundamental mechanisms of electron transport coupled phosphorylation. The existence of the PPi synthase is in line with the assumption that PPi may have preceded ATP as energy carrier between energy yielding and energy requiring reactions.     

Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls

Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated. Following chilling at 4[deg]C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures. Vacuolar ATPase levels did not change significantly throughout the chilling regime. However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ. Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein. Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.     

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.     

Partial characterization of H-translocating inorganic pyrophosphatase from 3 citrus varieties differing in vacuolar pH

Vacuolar pyrophosphatase (V-PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V-PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V-PPase activity. Samples from 'Valencia' orange had intermediate V-PPase levels. When assayed at equal V-PPase activity (measured as Pi production), V-PPase was not able to generate a pH gradient (ΔpH) in vesicles from acid lime, despite its capacity to form a ΔpH in the presence of ATP. Vesicles from sweet lime and 'Valencia' orange were able to form similar ΔpHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V-PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V-PPase polypeptide from mung bean recognized V-PPase from sweet lime and 'Valencia' orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton-pumping reactions of V-PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.     

Sequence analysis and transcriptional profiling of two vacuolar H+-pyrophosphatase isoforms in Vitis vinifera

Gene expression of grapevine vacuolar H(+)-pyrophosphatase (V-PPase EC 3.6.1.1.) during fruit ripening has previously been reported. Here we report on putative multiple V-PPase isoforms in grapevine. In this study a full-length cDNA sequence with an open reading frame of 2,295 nucleotides encoding a V-PPase gene (vpp2: acc. nr. AJ557256) was cloned. Sequence analyses of the deduced amino acid residues and RT-PCR experiments indicated that Vitis vinifera L. has at least two distinct isoforms of the V-PPase gene. Bioinformatic analyses of 13 V-PPase protein sequences revealed two highly conserved motifs associated with pyrophosphate (PPi) binding and response to stress, respectively. Both V-PPase isoforms were expressed at higher levels in the late post-véraison stage of grape berry ripening. Results also showed that the expression of grapevine V-PPase was induced by cold stress.     

Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum.

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.     

Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H⁺-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H⁺-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg²⁺ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H⁺-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO₃, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO₃ > KBr > K-acetate > K₂SO₄ > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H⁺-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H⁺ pumping as seen from the recovery of H⁺ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H⁺-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H⁺-ATPase to form a functional H⁺ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H⁺-ATPase in vivo.     

A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K_m for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective `PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

H+-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family.

The earliest known H+-proton-pumping inorganic pyrophosphatase, the integrally membrane-bound H+-proton-pumping inorganic pyrophosphate synthase from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-proton-pumping inorganic pyrophosphatase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-proton-pumping inorganic pyrophosphate synthase and two algal vacuolar H+-proton-pumping inorganic pyrophosphatases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-proton-pumping inorganic pyrophosphatases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-proton-pumping inorganic pyrophosphatases are reviewed and compared with H+-ATPases and soluble proton-pumping inorganic pyrophosphatases.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998