Single-strand DNA-scaffolded copper nanoclusters for the determination of inorganic pyrophosphatase activity and screening of its inhibitor.

A fluorescence method for the determination of inorganic pyrophosphatase (PPase) activity has been established based on copper nanoclusters (CuNCs). The polythymine of 40 mer (T40) acts as a template for the reduction reaction from Cu2+ to Cu0 by ascorbic acid (AA). This reaction leads to the formation of fluorescent CuNCs with excitation/emission peaks at 340/640 nm. However, the higher binding affinity between inorganic pyrophosphate (PPi) and Cu2+ hinders the effective formation of CuNCs. This shows low fluorescence intensity. PPase catalyzes the hydrolysis of PPi into Pi during which free Cu2+ ions are produced. This facilitates the formation of fluorescent CuNCs. Thus, the fluorescence intensity was restored. The fluorescence enhancement of the system has a linear relationship with PPase activity in the range 0.3 to 20 mU·mL-1, and the detection limit is0.2 mU·mL-1. The relative intensity (I/I0) at 640 nm for the analytical solution versus system is also employed to screen the inhibitor for PPase with high efficiency. Graphical abstract Schematic representation of a fluorescent assay for the determination of inorganic pyrophosphatase activity and screening its inhibitor based on single-strand polythymine-scaffolded copper nanoclusters.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Gold nanozyme as an excellent co-catalyst for enhancing the performance of a colorimetric and photothermal bioassay.

Advanced oxidation processes (AOPs) have recently proposed for advancing colorimetric sensing applications, owing to their excellent performance of sensitive color readout that generated from the oxidation of chromogenic substrates like 3,3',5,5'-tetramethylbenzidine (TMB) by reactive oxygen species (ROS) of AOPs such as ·OH and ·O2- radicals. However, the efficiency of ROS generation and the related H2O2 decomposition in most AOPs is quite low especially at neutral pH, which greatly hampered the practical sensing applications of the AOPs. We herein communicated that ß-cyclodextrin (ß-CD)-capped gold nanoparticles (ß-CD@AuNPs) can promote catalysis at neutral pH for AOP as an excellent co-catalyst. In this strategy, inorganic pyrophosphate (PPi) ions was first used to coordinate with Cu2+ and form Cu2+-PPi complex. In the presence of hydrogen peroxide, target inorganic pyrophosphatase (PPase) can hydrolyze PPi into inorganic phosphate (Pi) and release free Cu2+ simultaneously, resulting in a Cu2+-triggered Fenton-like AOP reaction. The introduced ß-CD@AuNPs acts as a co-catalyst, analogous to mediators in the most co-catalyzed system, to enhance the rate-limiting step of Cu2+/Cu+ conversion in Cu2+/H2O2 Fenton-like AOP and resulting in an efficient generation of ·OH and ·O2- radicals, which further producing an intense blue color by oxidizing TMB into its oxidation product (TMBox) within a short time. Finally, this reaction system was used to simply detecting target PPase with the colorimetric and photothermal readout based on the in-situ generated TMBox indicator. More significantly, we successfully demonstrated nanozyme can serve as a co-catalyst to promote the AOP catalysis at neutral pH, and inspire other strategies to overcome the pH limitation in the AOP catalysis and expand its colorimetric and photothermometric application.

A novel fluorometric method for inorganic pyrophosphatase detection based on G-quadruplex-thioflavin T.

In this paper, we propose a fluorometric approach for the highly sensitive detection of inorganic pyrophosphatase (PPase) based on G-quadruplex-thioflavin T (ThT). In the absence of PPase, Cu2+ can coordinate with pyrophosphate (PPi) to generate a Cu2+/PPi complex. Then the G-rich sequence folds into the G-quadruplex structure, which can combine with ThT to generate a remarkable fluorescent signal. In the presence of PPase, the coordinated compound can be destroyed by the PPase catalyzed hydrolysis of PPi into inorganic phosphate (Pi). The subsequent release of Cu2+ can compete with ThT to induce a tighter G-quadruplex structure, causing the release of ThT and a sharp fluorescence decrease. Based on this mechanism, a facile and quantitative strategy for PPase detection was developed. The fluorescence intensity of the system shows a linear relationship with the PPase activities in the range of 0.5-30 U/L with a detection limit as low as 0.48 U/L. The proposed strategy for fluorescence spectrometric PPase detection is convenient, cost effective, and sensitive. This can be utilized to evaluate the inhibition effect of NaF on PPase as well as diagnose PPase-related diseases.

Inhibition of Escherichia coli Inorganic Pyrophosphatase by Fructose-1-phosphate.

Pyrophosphate regulates vital cellular reactions, and its level in E. coli cells is under the ultimate control of inorganic pyrophosphatase. The mechanisms involved in the regulation of pyrophosphatase activity still need to be elucidated. The present study demonstrated that fructose-1-phosphate inhibits pyrophosphatase activity by a mechanism not involving competition with substrate for binding to the active site. The inhibition constant governing the binding of the inhibitor to the enzyme-substrate complex is 1.1 mM. Substitutions of Lys112, Lys115, Lys148, and Arg43 in the regulatory site completely or partially abolished the inhibition. Thus, Fru-1-P is a physiological inhibitor of pyrophosphatase that acts via a regulatory site in this enzyme.

Fluorescence biosensor for inorganic pyrophosphatase activity.

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PPi), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase). The best analogue (compound 16) with a Ki of 11 µM for MtPPiase is a species-specific inhibitor. Crystal structures of MtPPiase in complex with the lead compound and one of its analogues (compound 6) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric MtPPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PPi substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme-substrate complex. The inhibitors appear to allosterically lock the active site in a closed state causing its dysfunctionalization and blocking the hydrolysis. These inhibitors are the first examples of allosteric, species-selective inhibitors of PPiases, serving as a proof-of-principle that PPiases can be selectively targeted.

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus.

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3 (-) to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3 (-) was retained in roots of Xiangyou15. Moreover, NO3 (-) concentration in xylem sap, [(15)N] shoot:root (S:R) and [NO3 (-)] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3 (-) in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3 (-) long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3 (-) in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3 (-) allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 µM and 80 ± 8 µM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 µM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

A novel fluorescence assay for inorganic pyrophosphatase based on modulated aggregation of graphene quantum dots.

A simple and highly sensitive fluorometric method has been developed for inorganic pyrophosphatase (PPase) activity detection based on the disaggregation and aggregation of graphene quantum dots (GQDs). Copper ions can trigger the severe aggregation of GQDs with rich carboxyl groups, which results in effective fluorescence quenching. While, with the addition of pyrophosphate (PPi), the quenched fluorescence is effectively recovered owing to the strong interaction between PPi and Cu(2+). Furthermore, under the catalytic hydrolysis of PPase, the complex of PPi-Cu(2+)-PPi is rapidly disassembled, and the fluorescence is re-quenched. This method is highly sensitive and selective for PPase detection, with a linear correlation between the fluorescence intensity and the PPase concentration in the range from 1 to 200 mU mL(-1) with a detection limit down to 1 mU mL(-1) (S/N = 3). Additionally, the inhibition effect of NaF on the PPase activity is also studied. Thus, the proposed method may hold a potential application in the diagnosis of PPase-related diseases and screening of PPase inhibitors, to evaluate the function and inhibition of PPase in biological systems.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.

Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target.

The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Fluorometric method for inorganic pyrophosphatase activity detection and inhibitor screening based on click chemistry.

A fluorometric method for pyrophosphatase (PPase) activity detection was developed based on click chemistry. Cu(II) can coordinate with pyrophosphate (PPi), the addition of pyrophosphatase (PPase) into the above system can destroy the coordinate compound because PPase catalyzes the hydrolysis of PPi into inorganic phosphate and produces free Cu(II), and free Cu(II) can be reduced by sodium ascorbate (SA) to form Cu(I), which in turn initiates the ligating reaction between nonfluorescent 3-azidocoumarins and terminal alkynes to produce a highly fluorescent triazole complex, based on which, a simple and sensitive turn on fluorometric method for PPase can be developed. The fluorescence intensity of the system has a linear relationship with the logarithm of the PPase concentration in the range of 0.5 and 10 mU with a detection limit down to 0.2 mU (S/N = 3). This method is cost-effective and convenient without any labels or complicated operations. The proposed system was applied to screen the potential PPase inhibitor with high efficiency. The proposed method can be applied to diagnosis of PPase-related diseases.

Regulation of neurite growth by inorganic pyrophosphatase 1 via JNK dephosphorylation.

Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation.

A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.

Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase-pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z' = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.

Pyrophosphatase overexpression is associated with cell migration, invasion, and poor prognosis in gastric cancer.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate to form orthophosphate. Pyrophosphate can substitute for ATP under certain circumstances. We previously conducted a proteomic analysis to investigate tumor-specific protein expression in gastric cancer, and PPase was identified as a potential gastric tumor-specific marker; it was therefore selected for further study. Clinicopathological analysis, using proteomic analysis and immunohistochemistry, was used to validate PPase as a prognostic marker in gastric cancers. Proteomic analysis showed that PPase was overexpressed in patients with lymph node (LN) metastases and high tumor node metastasis (TNM) stages (p < 0.05). Based on immunohistochemistry, patients whose tumors overexpressed PPase had higher T stages, LN metastasis, a higher TNM stage, a higher cancer recurrence rate, and shorter survival times than patients whose tumors exhibited PPase underexpression (p < 0.05). Gain-of-function and loss-of-function approaches were employed to examine the malignant phenotypes of PPase-overexpressing or PPase-depleted cells. A decrease in PPase expression caused a significant decrease in gastric cancer cell migration and invasion in vitro, whereas forced overexpression of PPase enhanced migration but not invasion. Our findings indicate that PPase is involved in gastric tumor progression and that PPase may be a useful marker for poor prognosis of human gastric cancers.

[Alteration of transport activity of proton pumps in coleoptile cells during early development stages of maize seedlings].

Comparative analysis of the transport activity of proton pumps (plasmalemma H+-ATPase, vacuolar H+-ATPase, and vacuolar H+-pyrophosphatase) in the membrane preparations obtained from coleoptile cells ofetiolated maize seedlings (Zea mays L.) was carried out. The highest level ofvacuolar pyrophosphatase activity was observed during the early development of coleoptile cells under growth intensification through the elongation. The role of ATPase pumps of tonoplast and plasmalemma in the transport of hydrogen ions increases during further development. The plasmalemma activity in this process is higher. When the growth stops, the activity of proton pumps becomes significantly lower. Nevertheless, their substrate specificity and sensitivity to proton pump inhibitors do not change, which can be an evidence of physiological significance of pumps in the maintenance of cell homeostasis.

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.