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PubMed2000

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis.

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated K(eq)' (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25 degrees C is 110, and its standard-state free-energy change value (DeltaG') is -2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (K(m) = 0.5 mM). P(i) and phosphorylated compounds such as 3-phosphoglycerate, PP(i), ATP, ADP, NADP(+), and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-alpha-d-glucan 4-alpha-d-glucosyltransferase; EC) for ADPG, thus markedly blocking the starch biosynthesis.

Arabidopsis↗

PubMed2002

A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei.

Vacuolar proton pyrophosphatases (V-H(+)-PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H(+)-PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H(+)-PPase gene by using double-stranded RNA interference methodology to produce inducible V-H(+)-PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H(+)-PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 x 10(5) cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.

Adenosine Triphosphate↗

PubMed2002

Nudix hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5-phosphoribosyl 1-pyrophosphate (PRPP) pyrophosphatase activity that generates the glycolytic activator ribose 1,5-bisphosphate.

A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.

Binding Sites↗

PubMed2001

A plasma membrane-type Ca(2+)-ATPase co-localizes with a vacuolar H(+)-pyrophosphatase to acidocalcisomes of Toxoplasma gondii.

Ca(2+)-ATPases are likely to play critical roles in the biochemistry of Toxoplasma gondii, since these protozoa are obligate intracellular parasites and the Ca(2+) concentration in their intracellular location is three orders of magnitude lower than in the extracellular medium. Here, we report the cloning and sequencing of a gene encoding a plasma membrane-type Ca(2+)-ATPase (PMCA) of T.gondii (TgA1). The predicted protein (TgA1) exhibits 32-36% identity to vacuolar Ca(2+)-ATPases of Trypanosoma cruzi, Saccharomyces cerevisiae, Entamoeba histolytica and Dictyostelium discoideum. Sequencing of both cDNA and genomic DNA from T.gondii indicated that TgA1 contains two introns near the C-terminus. A hydropathy profile of the protein suggests 10 transmembrane domains. TgA1 suppresses the Ca(2+) hypersensitivity of a mutant of S.cerevisiae that has a defect in vacuolar Ca(2+) accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that TgA1 localizes to the plasma membrane and co-localizes with the vacuolar H(+)-pyrophosphatase to intracellular vacuoles identified morphologically and by X-ray microanalysis as the acidocalcisomes. This vacuolar-type Ca(2+)-ATPase could play an important role in Ca(2+) homeostasis in T.gondii.

Adenosine Triphosphate↗

PubMed2004

Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley.

Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop. Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots. The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10. In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression. These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses.

Amino Acid Sequence↗

PubMed1979

Interaction of a limited set of proteins with different mRNAs and protection of 5'-caps against pyrophosphatase digestion in initiation complexes.

A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.

Animals↗

PubMed1983

Substrate specificity of a nucleotide pyrophosphatase responsible for the breakdown of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from human placenta.

The membrane-bound enzyme responsible for the breakdown of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) has been purified 1,900-fold from detergent-solubilized human placenta, using chromatographies on Con A-Sepharose, Blue Sepharose, AMP-Agarose, and Sepharose CL-6B, and sucrose density gradient centrifugation. The enzyme required Mg2+ and showed the optimum activity at pH 9.4. The preparation was free of alkaline phosphatase [EC 3.1.3.1], phosphodiesterase [EC 3.1.4.1], and 5'-nucleotidase [EC 3.1.3.5] activities, which enabled investigation of the substrate specificity and kinetic properties of the enzyme without interference by secondary reactions due to the above activities. The enzyme cleaved the pyrophosphate linkages of NAD and various sugar nucleotides and the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate (PNTP), as well as the phosphosulfate linkages of PAPS and its biosynthetic precursor, adenosine 5'-phosphosulfate (APS), with apparent Km values of 0.12-0.33 mM. Relative activities towards PNTP and PAPS did not change during the purification procedures starting from the homogenate. This, together with the data of kinetic studies using two substrates simultaneously, led us to conclude that the activities towards all the substrates tested were due to one and the same nucleotide pyrophosphatase.

Female↗

PubMed1992

Common identity of substrate binding subunit of vacuolar h-translocating inorganic pyrophosphatase of higher plant cells.

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H(+)-translocating inorganic pyrophosphatase (tonoplast H(+)-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M(r)) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H(+)-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H(+)-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M(r(Beta)) = 64,500; M(r(Vigna)) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [(14)C]-N-ethylmaleimide. Similarly, the M(r) 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M(r) 66,000 subunit of the H(+)-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H(+)-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M(r) 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M(r) 64,500 to 67,000 in all four preparations. The M(r) 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M(r) 37,000 to 45,000 subunit pattern previously ascribed to the H(+)-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M(r) 66,000 subunit and the appearance of polypeptide fragments of M(r) 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H(+)-PPase has a common identity in all four organisms.

Journal Article↗

PubMed1992

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors.

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H(+)-PPase) from oat (Avena sativa L.) roots to changes in Mg(2+) and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H(+)-PPase is Mg(2)PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg(2+) and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H(+)-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H(+)-PPase. It is demonstrated that models incorporating Mg(2)PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H(+)-PPase. Calculations of the likely concentrations of Mg(2)PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H(+)-PPase would be about 70% saturated in vivo.

Journal Article↗

PubMed1992

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis.

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca(2+) and a number of other divalent cations (Co(2+), Mn(2+), Zn(2+)). By contrast, the H(+)-translocating ATPase (H(+)-ATPase) located on the same membrane is insensitive to Ca(2+). Here we examine the mechanism and feasibility of regulation of the vacuolar H(+)-PPase by cytosolic free Ca(2+) under the conditions thought to prevail in vivo with respect to Mg(2+), inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca(2+) or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg(2)PPi) at one site, inhibitory binding of Mg(2)PPi to a lower-affinity second site, binding of activator (Mg(2+)) at a third site, and direct binding of Ca(2+) or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca(2+) or CaPPi are explicable only if Ca(2+), rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca(2+) on the substrate concentration dependence of H(+)-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H(+)-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca(2+) might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.

Journal Article↗

PubMed1994

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases.

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

Journal Article↗

PubMed1994

Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds.

Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings.

Journal Article↗

PubMed1995

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.

Journal Article↗

PubMed1996

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors).

Inhibitors preferentially and covalently reactive with cysteine, arginine, histidine, and carboxyl-containing residues were inhibitory to the plant vacuolar H+-transporting inorganic pyrophosphatase (H+-PPase) from Vigna radiata (mung bean) and Beta vulgaris (red beet), but hydrophobic compounds and those reactive with tyrosine and lysine were less effective. Inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, and N-ethylmaleimide was decreased in the presence of Mg2+ or mixtures of Mg2+ and inorganic pyrophosphate (PPi) but not by PPi alone. None of these ligands affected inhibition by reagents reactive with histidine. The Mg2+ dependence of protection from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide inhibition followed first-order kinetics and yielded a Km for free Mg2+ of 20 to 23 [mu]M. Protection from inhibition by N-ethylmaleimide and phenylglyoxal varied as a function of Mg2PPi concentration, suggesting that this is the substrate for the H+-PPase. Protection by Mg2PPi followed Michaelis-Menten kinetics with a Km of approximately 2 [mu]M. These results are consistent with the predictions of a kinetic model for the H+-PPase (R.A. Leigh, A.J. Pope, I.R. Jennings, D. Sanders [1992] Plant Physiol 100: 1698-1750), which identified free Mg2+ as an allosteric activator (Km = 25 [mu]M) and Mg2PPi as the substrate (Km = 2.5-5 [mu]M).

Journal Article↗

PubMed1997

Tris Is a Competitive Inhibitor of K+ Activation of the Vacuolar H+-Pumping Pyrophosphatase.

The effects of a range of commonly used pH buffers on the hydrolytic activity of the plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase) from mung bean (Vigna radiata L.) hypocotyls were tested. All of the buffers inhibited K+ stimulation of the V-PPase, and the degree of inhibition was dependent on the concentrations of both the buffer and K+. The effects were dependent on the organic cation used in the buffers, and those tested inhibited in the order: Tris > Bis-Tris-propane > Bicine = Tricine > imidazole. Detailed studies revealed that a model in which Tris affects both the Km and Vmax for K+ stimulation provided an accurate description of the observed kinetics. The ability of different cations to stimulate the V-PPase was measured with a noncompeting buffer (5 mM imidazole-HCl) and the order of effectiveness was K+ = Rb+ > NH4+ >> Cs+ > Na+ > Li+, with the Km for K+ stimulation being about 1 to 2 mM. Published experiments performed in the presence of Tris were re-evaluated and all could be fitted to mixed inhibition kinetics, with kinetic parameters similar to those measured for the mung bean V-PPase. It is concluded that the variations in the published Km for K+ stimulation of the V-PPase are probably due to the effects of pH buffer cations and that the real value for this parameter is in the low millimolar range. The implications of this for regulation of the V-PPase by K+ in vivo and for the role of the enzyme in K+ transport into the vacuole are discussed.

Journal Article↗

PubMed1998

Reversibility of H+-ATPase and H+-pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to 32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.

Journal Article↗

PubMed1985

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles.

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl(-) and inhibited by NO(3) (-). NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K(2)SO(4) supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K(+). KCl and K(2)SO(4) support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.

Journal Article↗

PubMed1986

Chromatographic resolution of h-translocating pyrophosphatase from h-translocating ATPase of higher plant tonoplast.

Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl(-)-stimulated, NO(3) (-)-inhibited ATPase, and a K(+)-stimulated, Na(+)-inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Triton X-100 containing 4 millimolar MgCl(2) and 1 millimolar ethylenediamine tetracetic acid, as protectants, gives high yields of both the ATPase and PPase in soluble form. Chromatography of the solubilized membranes on Sephacryl-400 results in the separation of the two enzymes. The PPase and ATPase are purified 4- and 17-fold, respectively, with quantitative recovery. The separated enzymes show negligible activity towards the other's substrate and the separated PPase only hydrolyzes pyrophosphate. The separated enzymes show mineral ion requirements identical to those of the corresponding pump and hydrolytic activities in native tonoplast and both solubilized enzymes are subject to phospholipid activation.

Journal Article↗