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PubMed1989

Ca2+-induced accumulation of pyrophosphate in mitochondria during acetate metabolism.

The mechanism of pyrophosphate (PPi) accumulation in rat liver during acetate metabolism was investigated. Perfusion of the liver with acetate in the presence of noradrenaline and glucagon induced marked accumulation of PPi (2 mumol/g of liver, 200 times that of control). In contrast, perfusion with glutamine, which generates PPi only in the cytosol, caused little accumulation of PPi, even in the presence of the two hormones. The site of PPi accumulation was shown to be the mitochondria by the finding that isolated mitochondria from the liver perfused with acetate and the hormones contained 50 nmol of PPi/mg of protein. The addition of an uncoupler to mitochondria with accumulated PPi caused gradual decrease in their PPi content, with concomitant release of a stoichiometric amount of Ca2+. Similar accumulation of PPi was observed when isolated mitochondria were incubated with acetate and Ca2+. These results show that an increase in cytosolic Ca2+ caused by the co-administration of the two hormones induced uptake of the ion into mitochondria, and that PPi accumulated in mitochondria only when it was generated in the organelles with an elevated concentration of Ca2+. High mitochondrial concentrations of Ca2+ are considered to inhibit inorganic pyrophosphatase through the formation of a stable complex, CaPPi-. Mitochondria with accumulated PPi had normal respiratory activities, and their adenine nucleotide concentrations were increased 2-fold rather than being decreased, the increases also being considered to be caused by their high concentration of Ca2+.

Acetates↗
PubMed1993

The effect of thiamin on the activation of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in Euglena gracilis.

The effect of thiamin on thiamin pyrophosphate-dependent 2-oxoglutarate (2-OG) decarboxylase activity in Euglena gracilis was investigated. The total activity of 2-OG decarboxylase in thiamin-sufficient cells in 3 times that in thiamin-deficient cells. The addition of thiamin to thiamin-deficient cells causes the total enzyme and holoenzyme activities to increase and reach similar levels to that in thiamin-sufficient cells. Cycloheximide and chloramphenicol, inhibitors of protein synthesis, have no effect on the total enzyme activity. Immunochemical titration and determination of 2-OG decarboxylase mRNA by using an antibody directed against Euglena 2-OG decarboxylase indicate that the increase in the holoenzyme activity of 2-OG decarboxylase is due to activation of pre-existing protein and does not require synthesis of new proteins in thiamin-deficient cells. During the period of the increase in the total activity, the apoenzyme increases and reaches a temporary peak in 2 h. Immunoblot analysis demonstrates that the precursor form (a 65 kDa subunit) of 2-OG decarboxylase in thiamin-deficient cells is more abundant than that in thiamin-sufficient cells and the increase in the apoenzyme by addition of thiamin results from the conversion of the precursor form into the mature form (a 62 kDa subunit).

Animals↗
PubMed1976

The influence of orthophosphate on the renal handling of inorganic pyrophosphate in man and dog.

1. The urinary excretion of inorganic pyrophosphate (PPi), a known inhibitor of the growth and aggregation of crystals of calcium phosphate and calcium oxalate, increases after ingestion of orthophosphate (Pi). This effect may contribute to the apparent ability of oral phosphate to reduce the formation of urinary stones in man. This paper is a study of the mechanism by which Pi increases PPi excretion, investigated by renal clearance techniques in man and renal arterial infusion in dogs. PPi in plasma was measured by an isotope-dilution method after ion-exchange chromatography. 2. The mean renal clearance of endogenous PPi in ten men was 7-9 +/- 1-7 (SE) ml/min, and the mean ratio of PPi clearance to creatinine clearance was 0-08 +/- 0-02 (SE). The oral ingestion of Pi increased the urinary excretion and renal clearance of PPi about threefold, without significantly changing its concentration in plasma. 3. In dogs, the infusion of Pi into one renal artery caused a greater increase in urinary PPi from the infused than from the non-infused kidney, an effect that could be accentuated by simultaneous intravenous infusion of PPi. In dogs, only 1-3% of an injected or infused dose of PPi appeared intact in the urine, regardless of whether it was infused into the systemic or renal circulation. 4. These results suggest that Pi has a direct affect on the kidney to increase the excretion of PPi. It is possible that Pi either interferes with tubular reabsorption of PPi, perhaps by competing for a common tubular transport mechanism, or that Pi diminishes the intrarenal hydrolysis of PPi.

Animals↗
PubMed1978

Uptake by bone of pyrophosphate, diphosphonates and their technetium derivatives.

1. The uptake of inorganic pyrophosphate (PPi) from blood to bone was investigated in the rat in vivo. 2. PPi is taken up by the bone, where it appears both as PPi and as inorganic orthophosphate (Pi). The latter is due at least partly to local hydrolysis. 3. The fraction of injected PPi taken up by bone, measured as total PPi, was in the same range as that of technetium-tin-PPi, diphosphonates, technetium-tin-ethane-1-hydroxy-1,1-diphosphonate and Pi, but lower than that of calcium. 4. The plasma half-life of PPi is in the same order of magnitude as that of technetium-tin-PPi, diphosphonates, technetium-tin-ethane-1-hydroxy-1,1-diphosphonate, Pi and calcium. 5. PPi, diphosphonates and their technetium complexes are only partly ultrafiltrable in plasma. 6. It appears that the technetium complexes behave in a similar fashion to free PPi or diphosphonate.

Animals↗
PubMed2000

Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate.

In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32P-PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell-suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.

Animals↗
PubMed2001

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation.

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Diphosphates↗
PubMed2002

Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.

Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.

Adenine Nucleotides↗
PubMed2000

Differential diagnosis of calcium pyrophosphate dihydrate deposition of the temporomandibular joint.

Calcium pyrophosphate dihydrate (CPPD) deposition disease (pseudogout) of the temporomandibular joint (TMJ) is rare. It is characterized by the presence of crystal deposits that are birefringent under polarized light. Although these crystals are characteristically weakly birefringent, some other crystals such as those of calcium oxalate, synthetic steroids, and ethylenediaminetetraacetic acid are also birefringent. The differential diagnosis should therefore be based on a quantitative analysis of crystals or observation of the crystal structure in calcified sections. We present a case of CPPD deposition disease of the TMJ and report on the value of such an analysis to substantiate the diagnosis.

Biopsy↗
PubMed1975

[A comparison of radiology and scanning with 99mTc-pyrophosphate in 125 patients with tumours (author's transl)].

Various portions of the skeleton were examined in 125 patients both radiologically and by scanning, using 99mTc-pyrophosphate. The results are compared, particular attention being paid to errors in diagnosis. The figures show that scans represent a valuable additional form of examination for detecting tumours which have not been found radiologically. The importance of serial observations is discussed.

Bone Neoplasms↗
PubMed2001

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate.

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP(4)) from InsP(5). Additionally, mIPMK forms InsP(4) from Ins(1,4,5)P(3) and InsP(5) from Ins(1,3,4,5)P(4).

Amino Acid Sequence↗
PubMed1976

Differential synthesis of blocked and unblocked 5'-termini in reovirus mRNA: effect of pyrophosphate and pyrophosphatase.

Viral mRNA synthesized in vitro by reovirusassociated enzymes contained 5'-terminal ppG, GpppG, or m7GpppGm. Selective synthesis of one type of 5'-end was obtained by adjusting the composition of the transcription reaction mixture based on a series of reactions proposed for formation of blocked, methylated 5'-terminal sequences of mRNA (capformation). The presence of inorganic pyrophosphate increased the proportion of ppG 5'-ends, while addition of pyrophosphatase yielded mRNA with 5'-terminal GpppG, which in the presence of a methyl donor was converted to m7GpppGm.

Diphosphates↗
PubMed1992

Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.

The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H+ pumps: a vacuolar ("V-type") H(+)-ATPase (EC 3.6.1.3) and a H(+)-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1). The H(+)-PPase, like the V-type H(+)-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast H(+)-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H(+)-PPase of Arabidopsis thaliana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H(+)-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the Mr 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H(+)-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H(+)-PPase and any other characterized H+ pump or PPi-dependent enzyme implies a different evolutionary origin for this translocase.

Amino Acid Sequence↗
PubMed1995

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.

Adult↗
PubMed1995

Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels.

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.

Adenosine Triphosphate↗
PubMed1995

Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment).

We have employed site-directed mutagenesis to identify those amino acid residues that interact with the deoxynucleoside triphosphate (dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary complex. Earlier structural, mutagenesis, and labeling studies have suggested that the incoming dNTP molecule contacts a region on one side of the polymerase cleft, primarily involving residues within the so-called "fingers" subdomain. We have made mutations in residues seen to be close to the dNTP in the crystal structure of the Klenow fragment-dNTP binary complex and have examined their kinetic parameters, particularly Km(dNTP). The results are consistent with the notion that there are significant differences between the dNTP interactions in the binary and ternary complexes, although some contacts may be present in both. When dTTP is the incoming nucleotide, the side chains of Arg754 and Phe762 make the largest contributions to binding; measurement of Km(PPi) suggests that Arg754 contacts the beta- or gamma-phosphate of the dNTP. With dGTP, the contribution of Arg754 remains the same, but the additional interactions are provided by both Lys758 and Phe762, suggesting that the binding of the incoming dNTP is not identical under all circumstances. Mutations in Arg754 and Lys758 also cause a substantial decrease in the rate of polymerase-catalyzed incorporation, and sulfur elemental effect measurements indicate that loss of Arg754 (and perhaps also Lys758) slows the rate of the chemical step of the reaction. Mutations of Arg682, His734, and Tyr766 affect the binding of DNA, suggesting that these mutations, whose effect on dNTP binding is small, may influence dNTP binding indirectly via the positioning of the DNA template-primer.

Amino Acid Sequence↗
PubMed1996

The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity.

We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity. DGPP phosphatase activity was absent in pgpB mutant cells and was expressed at high levels in cells carrying the wild-type pgpB gene on a runaway replication plasmid. The pgpB mutant has been primarily characterized by a defect in phosphatidate (PA) phosphatase activity and also exhibits defects in lyso-PA phosphatase and phosphatidylglycerophosphate phosphatase activities. The defective PA phosphatase in the pgpB mutant was shown to be a Mg2+-independent PA phosphatase activity of the DGPP phosphatase enzyme. We characterized DGPP phosphatase activity in membranes from cells overproducing the pgpB gene product. DGPP phosphatase catalyzed the dephosphorylation of the beta phosphate of DGPP to form PA followed by the dephosphorylation of PA to form diacylglycerol. The specificity constant (Vmax/Km) for DGPP was 9.3-fold greater than that for PA. The pH optimum for the DGPP phosphatase reaction was 6. 5. Activity was independent of a divalent cation requirement, was potently inhibited by Mn2+ ions, and was insensitive to inhibition by N-ethylmaleimide. Pure DGPP phosphatase from Saccharomyces cerevisiae was shown to be similar to the E. coli DGPP phosphatase in its ability to utilize lyso-PA and phosphatidylglycerophosphate as substrates in vitro.

Cell Membrane↗
PubMed1997

Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity.

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

Amino Acid Sequence↗
PubMed1997

Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide.

On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane alpha-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thaliana were singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229 (227-245) of the V-PPase and the N,N'-dicyclohexylcarbodiimide (DCCD)-binding transmembrane alpha-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y. , and Strid, A. (1993) Plant Cell Physiol. 34, 375-378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+ translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+ translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+ translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides.

Amino Acid Sequence↗