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PubMed1999

Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.

Bisphosphonates, synthetic compounds which suppress bone resorption, are used in the treatment of skeletal disorders. Their mode of action and intracellular targets have not yet been identified. Recent evidence suggested that enzymes of the mevalonate pathway are the potential targets. In this study, we examined the effect of four potent nitrogen (N)-containing bisphosphonates, clodronate and NH2-olpadronate, an inactive analogue of olpadronate, on isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and protein geranylgeranyl transferase I activity. We found that all N-containing bisphosphonates inhibited isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity dose dependently with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo, whereas clodronate and NH2-olpadronate had no effect. Furthermore, none of the bisphosphonates tested affected geranylgeranyl pyrophosphate synthase or geranylgeranyl transferase I activity. Our study reveals for the first time the intracellular target of N-containing bisphosphonates and supports the view that all bisphosphonates do not share the same molecular mechanism of action.

Animals↗
PubMed1989

Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate.

The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose-6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration.

Journal Article↗
PubMed1990

Isolation and sequence of the human farnesyl pyrophosphate synthetase cDNA. Coordinate regulation of the mRNAs for farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and 3-hydroxy-3-methylglutaryl coenzyme A synthase by phorbol ester.

We report the isolation and nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA, an enzyme in the cholesterogenic pathway. Partial cDNAs for the human farnesyl pyrophosphate synthetase were isolated by screening human hepatoma (HepG2) and placental cDNA libraries with the rat liver cDNA for farnesyl pyrophosphate synthetase as a probe. Anchored polymerase chain reaction was used to isolate the 5'-end of the cDNA. The nucleotide sequence of the human farnesyl pyrophosphate synthetase cDNA has high identity (86%) to the rat liver cDNA. Treatment of the human monocytic leukemia cell line THP-1 with phorbol esters led to 2--7-fold increases in mRNA concentrations for the three cholesterogenic enzymes, farnesyl pyrophosphate synthetase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and HMG-CoA synthase within 5 h. Immunoprecipitation of radiolabeled cells demonstrated that there was a corresponding increase in the rate of synthesis of all three proteins. The addition of cycloheximide to cells also led to increases in the mRNA concentrations of the three enzymes. Treatment of cells with phorbol esters and cycloheximide resulted in superinduction of all three mRNAs; HMG-CoA synthase mRNA levels increased 35-fold, farnesyl pyrophosphate synthetase 17-fold, and HMG-CoA reductase 16-fold 5 h after treatment. The mRNA levels returned to pretreatment levels by 20 h. Cells were also preincubated in the presence of a lipoprotein-deficient fraction of serum plus mevinolin to induce the levels of the three mRNAs. Addition of phorbol esters and cycloheximide to these derepressed cells led to further increases in the mRNA levels for all three enzymes. These results are consistent with the hypothesis that THP-1 cells contain a short-lived negative transcription factor which regulates transcription of the FPP synthetase, HMG-CoA reductase, and HMG-CoA synthase genes. Phorbol esters also regulate these same genes, presumably by modifying a common negative transcription factor and/or by inducing a positive transcription factor(s).

Amino Acid Sequence↗
PubMed1983

Pyrophosphate, phosphate ion interaction: effects on calcium pyrophosphate and calcium hydroxyapatite crystal formation in aqueous solutions.

The relationship between ambient ionic conditions that favor pyrophosphate (PPi) versus phosphate (Pi) biomineralization is important to understanding the pathogenesis of chondrocalcinosis. We studied aqueous solutions at pH 7.4, 37 degrees C, [Na+] = 140 mM, [Mg+ +] = 0.5 mM, [Ca+ +] = 1.0 or 1.5 mM over a range of pyrophosphate and phosphate concentrations to determine the effect of different ambient concentrations and ratios of Pi/PPi on calcium pyrophosphate dihydrate (CPPD) and calcium hydroxyapatite (HA) crystal formation. We found that the Pi/PPi ratio is an extremely important determinant of the crystal product formed. At low [Pi], CPPD crystal formation is partially inhibited by Pi; at higher [Pi], calcium pyrophosphate, calcium phosphate and calcium pyrophosphate-phosphate complexes amorphous to x-ray diffraction are formed; whereas at still higher [Pi], HA crystal formation partially inhibited by PPi. We conclude that CPPD forms when the ratio [Pi]/[PPi] less than 3 and HA forms when [Pi]/[PPi] greater than 100.

Calcium Pyrophosphate↗
PubMed1999

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs.

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.

Animals↗
PubMed1985

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase of Rhodospirillum rubrum: effects of Mg2+, phosphate, and pyrophosphate.

The relation that exists between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for added Mg2+. Optimal rates of hydrolysis were attained at 1 mM Mg2+ with 0.67 mM pyrophosphate; the rate od hydrolysis correlated with the concentration of Mg-pyrophosphate, which indicated that the latter was the substrate for hydrolysis. The Pi-PPi exchange reaction rate was low at concentrations of added Mg2+ below 1 mM (0.67 mM pyrophosphate), but increased as the concentration of Mg2+ in the medium was increased. The Pi-PPi exchange reaction depends on the concentration of MgHPO4, which suggests that this is the substrate in the exchange reaction. However, it is likely that free Mg2+ also exerts a favorable effect on the Pi-PPi exchange reaction. The optimal concentration for the Pi-PPI exchange reaction was approx 240 microM, which suggests that the concentration of the hydrolyzable substrates modulates the kinetic characteristics of the enzyme.

Cell Membrane↗
PubMed1981

Isoleucyl-tRNA synthetase from Baker's yeast. Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate.

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Adenosine Triphosphate↗
PubMed1992

Urinary excretion of inorganic pyrophosphate by normal subjects and patients with renal calculi in north-western India and the effect of diclofenac sodium upon urinary excretion of pyrophosphate in stone formers.

24 h urinary pyrophosphate excretion was studied in 20 normal healthy subjects and 75 idiopathic stone formers from north-western regions of India. The mean 24-hour urinary excretion of pyrophosphate was significantly low in stone formers (50.67 +/- 2.16 mumol/24 h) as compared to that of normal subjects (71.46 +/- 5.46 mumol/24 h) (p less than 0.01). Diclofenac sodium, a non-steroidal anti-inflammatory agent, was administered 50 mg thrice daily for 1 week to 18 stone formers and 24-hour urinary pyrophosphate excretion was studied before and after drug therapy. The 24-hour urinary excretion of pyrophosphate increased from 54.32 +/- 21.40 to 78.31 +/- 28.03 mumol subsequent to diclofenac sodium therapy (p less than 0.01).

Adult↗
PubMed1979

Quantification of human plasma inorganic pyrophosphate. I. Normal values in osteoarthritis and calcium pyrophosphate dihydrate crystal deposition disease.

The methodologic variables of the UDPG pyrophosphorylase method for analysis of inorganic pyrophosphate (PPi) levels in biologic fluids are described. Use of a tourniquet in collection of blood specimens elevated plasma PPi levels from 35% to 55% above control values and may explain the differences in published normal values. The sodium pyrophosphate decahydrate used to prepare the standard solution lost 8 waters of hydration after dessication, which could result in the calculation of spuriously elevated PPi levels. Normal plasma PPi concentration was 2.18 muM with a range (95% confidence limits) of 0.58-3.78 muM. Comparison of plasma PPi in normal subjects, patients with primary osteoarthritis, and patients with calcium pyrophosphate dihydrate deposition disease revealed no significant intergroup differences.

Blood Specimen Collection↗
PubMed2000

Zinc(II) and cadmium(II) metal complexes of thiamine pyrophosphate and 2-(alpha-hydroxyethyl)thiamine pyrophosphate: models for activation of pyruvate decarboxylase.

Metal complexes of thiamine pyrophosphate (TPP) of the general formula [M2(TPPH)2Cl2]x4H2O (M = Zn2+, Cd2+) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(alpha-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed.

Apoenzymes↗
PubMed1986

Isopentenyl pyrophosphate isomerase:dimethylallyl pyrophosphate isomerase: isolation from Claviceps sp. SD 58 and comparison to the mammalian enzyme.

Isopentenyl pyrophosphate isomerase:dimethyl pyrophosphate isomerase (EC 5.3.3.2) has been purified to near homogeneity from Claviceps sp. A molecular weight of 35,000 was found by gel exclusion chromatography as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the enzyme consists of a single subunit and is in contrast to the Mr 22,000 that we have found for the enzyme from liver. The lability of isomerase from liver, often reported, has been found to be due to its susceptibility to proteolysis. Nine compounds have been tested as inhibitors of both isomerases. The binding of analogs requires the pyrophosphate moiety which may be substituted by a variety of alkyl groups. Inclusion of a polar function in the hydrocarbon portion of the analog greatly reduces interaction with the enzyme. Reversibility of the reaction was not found with a higher homolog of the substrate.

Animals↗
PubMed1999

Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants.

Cell cultures of Catharanthus roseus were supplied with [2-13C, 3-2H]-deoxyxylulose or [2-13C,4-2H]1-deoxyxylulose. Lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR and 1H,2H-decoupled 13C NMR. From the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect to the internal 13C label). The detected stereochemical signature implies that the label is located preferentially in the (E)-hydrogen atom of IPP. This preferential labeling, in turn, rules out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In the experiment with [2-13C, 4-2H]1-deoxyxylulose, the 13C label was efficiently transferred to the terpenoids whereas the 2H label was completely washed out, most probably after IPP formation as a consequence of the isomerization and elongation process. In addition, the data cast light on the stereochemical course of the dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

Journal Article↗
PubMed1977

Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate.

A procedure was devised to detect and assay uridine 5'-pyrophosphate (UDP)-glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP-glucuronic acid and (32)P-pyrophosphate, and the (32)P-uridine 5'-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine (32)P-adenosine 5'-triphosphate produced by adenosine 5'-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5'-triphosphate.

Journal Article↗
PubMed1991

Inorganic pyrophosphate in metabolic diseases predisposing to calcium pyrophosphate dihydrate crystal deposition.

Inorganic pyrophosphate (PPi) levels were estimated by radiometric assay in urine and in synovial fluid (SF) from asymptomatic, nonarthritic knees of patients with untreated metabolic disease and normal controls. SF PPi was significantly elevated in patients with hyperparathyroidism (mean +/- SEM 19 +/- 3 microM; n = 9), hemochromatosis (23 +/- 5 microM; n = 6), and hypomagnesemia (27 +/- 0.1 microM; n = 2) compared with normal subjects (10 +/- 0.5 microM, n = 50), and was low in patients with hypothyroidism (4.2 +/- 2.3 microM; n = 11) (P less than 0.05 all comparisons). Urinary PPi was elevated only in those with hypophosphatasia. Local elevation of ionic PPi may be relevant to the mechanism of crystal formation in metabolic diseases predisposing to calcium pyrophosphate dihydrate (CPPD) crystal deposition. The finding of low SF PPi levels in patients with hypothyroidism further questions the association between this condition and CPPD.

Adult↗
PubMed1986

Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease.

In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality.

Calcium Pyrophosphate↗
PubMed1995

Mapping of the pyrophosphate binding sites of beef heart mitochondrial F1-ATPase by photolabelling with azidonitrophenyl [alpha-32P]pyrophosphate.

4-Azido-2-nitrophenyl [alpha-32P]pyrophosphate (azido-[alpha-32P]PPi) mimics ADP and PPi by some of its binding properties when assayed in the absence of photoirradiation with mitochondrial F1-ATPase. Upon photoirradiation, both alpha- and beta-subunits of F1-ATPase were covalently labelled. Following chemical and enzymatic cleavages of each of the two photolabelled subunits, peptides containing the covalently bound radioactivity were separated by HPLC and identified by amino acid sequencing. Bound azido-[alpha-32P]PPi was found to be concentrated in two distant sequences of the alpha-subunit, namely Asp194-Thr221 and Lys386-Met437, and in a single sequence of the beta-subunit Glu294-Met358 with most of the photoprobe bound to beta-Tyr-311 and beta-Tyr-345. These results are discussed in terms of a model in which the pyrophosphate binding sites of F1 are located in regions of the alpha- and beta-subunits exposed at the interface between the two subunits and correspond to non-catalytic and catalytic adenine nucleotide binding sites, respectively.

Affinity Labels↗
PubMed1983

Essential protein factors for polyprenyl pyrophosphate synthetases. Separation of heptaprenyl pyrophosphate synthetase into two components.

Heptaprenyl pyrophosphate synthetase from Bacillus subtilis was dissociated into two essential components, none of which had catalytic activity alone. The enzyme activity was restored when the two components were combined with each other. Both fractions, designated components I and II in the order of their elution from DEAE-Sephadex, appeared to be proteins of Mr 30000. Component I was much more stable than component II which was easily destroyed by relatively mild heat treatment. Neither was interchangeable with any of the essential components of hexaprenyl pyrophosphate synthetase of Micrococcus luteus B-P 26.

Alkyl and Aryl Transferases↗
PubMed1998

A pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum.

The integrally membrane-bound, proton-pumping inorganic pyrophosphate (PPi) synthase in phototrophic bacteria is hitherto the only described alternative to the ATP synthase in biological electron transport phosphorylation. We have identified and sequenced the first gene coding for a pyrophosphate synthase. The deduced protein contains 660 amino acid residues and 15 putative membrane-spanning segments. It is homologous to the vacuolar pyrophosphatases from plants.

Amino Acid Sequence↗