Dimeric structure of H-translocating pyrophosphatase from pumpkin vacuolar membranes

Vacuolar membrane H⁺-translocating pyrophosphatase (H⁺-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H⁺-PPase [(1989) J. Biol. Chem. 264, 20068–20073]. The native, functional molecular size of the pumpkin H⁺-PPase was estimated to be 135–139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H⁺-PPase, and also probably H⁺-PPases from other plants, is a dimer of 75 kDa subunits.     

Oxygen exchange reactions catalyzed by vacuolar H-translocating pyrophosphatase Evidence for reversible formation of enzyme-bound pyrophosphate

Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H⁺-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate P_i---HOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases.     

Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.     

Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus.

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly.     

Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans.

An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

Reconstitution of transport function of vacuolar H(+)-translocating inorganic pyrophosphatase.

A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Primary structure of the inorganic pyrophosphatase from thermophilic bacterium PS-3

The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes.     

Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris

A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2+, and the (Mg-PPi) complex is regarded to be the functional substrate. Free Mg2+ revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2+. Mg2+ is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2+ revealed a non-competitive type of inhibition on the Mg2+-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2+, had to be applied. An enrichment method along with stepwise lowering the concentrations of Triton X-100 and Mg2+ after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2+ was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity.     

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 [¹⁴C]-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.     

Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.     

Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus.

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.     

Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3

A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).     

Alkaline inorganic pyrophosphatase from immature wheat grains

Changes in the level of alkaline inorganic pyrophosphatase and ADPG-pyrophosphorylase were monitored in developing wheat grains at weekly intervals aft     

Purification of inorganic pyrophosphatase from rat salivary glands

Inorganic pyrophosphatase [EC 3.6.1.1] isolated from rat sublingual and submandibular glands was purified 2300-fold and 2600-fold, respectively. The purified enzymatic preparations separated on electrophoresis into two protein bands, of which only one showed the pyrophosphatase activity. Inorganic pyrophosphatase from rat salivary glands is a monomeric anionic protein, its isoelectric point is 5.42 and 4.90 for the sublingual and submandibular glands, respectively.     

Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.