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PubMed1983

Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate.

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed alpha-pinene, beta-pinene, limonene, and gamma-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn2+. It could be replaced with about 2% effectiveness by Mg2+ and Co2+. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn2+)2 species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.

Alkyl and Aryl Transferases↗
PubMed1988

Monoterpene cyclases: physicochemical features required for pyrophosphate binding determined from inhibition by structural analogs.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate:(+)-alpha-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage (Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.

Binding, Competitive↗
PubMed1989

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes.

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.

Animals↗
PubMed1991

Pyrophosphate inhibition of Proteus mirabilis-induced struvite crystallization in vitro.

Struvite (MgNH4PO4.6H2O) crystals, the major mineral component of infectious urinary calculi, were produced in vitro by growth of a clinical isolate of Proteus mirabilis in artificial urine. P. mirabilis growth and urease-induced struvite production were monitored by phase contrast light microscopy and measurements of urease activity, pH, ammonia concentrations, turbidity, and culture viability. In the absence of pyrophosphate, struvite crystals appeared within 3-5 h due to the urease-induced elevation of pH and initially assumed a planar or 'X-shaped' crystal habit (morphology) characteristic of rapid growth. When pyrophosphate was present, initial precipitation and crystal appearance were significantly impaired and precipitates were largely amorphous. When crystals did appear (usually after 7 or 8 h) they were misshapen or octahedral in shape indicative of very slow growth. X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) identified all crystals as struvite. Trace contaminates of carbonate-apatite (Ca10(PO4)6CO3) or newberyite (MgHPO4.H2O) were produced only in the absence of pyrophosphate. P. mirabilis viability and culture pH elevation were unaffected by the addition of pyrophosphate, whereas urease activity and ammonia concentrations were marginally reduced. Struvite could also be produced chemically by titration of the artificial urine with NH4OH. If pyrophosphate was present during titration, the same inhibitory effect on crystal growth occurred, so it is unlikely that urease inhibition is important. Lowering of pyrophosphate concentration from 13-0.45 mumol/l did not reduce its inhibitory activity so it is unlikely to act by chelating free Mg2+. We propose that pyrophosphate inhibits struvite growth principally through direct interference with the chemical mechanisms involved in crystal nucleation and growth, because of its effectiveness at very low concentrations.

Ammonia↗
PubMed1977

Usefulness of preoperative and postoperative Tc-99m (Sn)-pyrophosphate scans in patients with ischemic and valvular heart disease.

To assess the usefulness of myocardial imaging with technetium-99m-stannous pyrophosphate for detecting acute myocardial necrosis in patients undergoind cardiac surgery, 66 such patients were stldied. Tc-99m (Sn)-pyrophosphate scans were obtained in all patients 3 to 6 days postoperatively and in 45 preoperatively. Electrocardiograms and serum samples for measuring myocardial isoenzyme of creatine kinase (MB CK) levels were obtained before and serially after cardiac surgery. Seven of the 46 patients undergoing myocardial revascularization had a definite new myocardial infarction as indicated by electrocardiogram and MB CK isoenzyme concentrations, and postoperative pyrophosphate scans were abnormal in all but one. In addition, six of the eight patients with possible myocardial infarction (elevated MB CK levels and persistent ST-T wave depressions) had an abnormal scan postoperatively. Seven of the 20 patients undergoing aortic or mitral valve replacement, or both, had a possible postoperative myocardial infarction by electrocardiogram and MB CK criteria and the myocardial scan was positive in two. All the patients with a normal electrocardiogram and normal MB CK levels had a normal pyrophosphate scan. Preoperative scans were obtained in 22 patients wit; valvular heart disease and were positive in two with a heavy calcified mitral valve on fluoroscopy and in one with a calcified aortic valve. After valve replacement, the pyrophosphate scan became normal in two patients and remained abnormal in the third patient with electrocardiograms and MB CK levels suggesting acute myocardial infarction. We conclude that the Tc-99m (Sn)-pyrophosphate scan is useful for analyzing the occurrence of acute myocardial infarction in patients undergoing cardiac surgery and that, in conjunction with the electrocardiogram, it permits confirmation or exclusion of that diagnosis. Furthermore, false positive pyrophosphate scans may occur in patients with heavy valve calcifications.

Adult↗
PubMed2002

Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s(-1)) was 100 times larger than the steady-state rate (0.02 s(-1)). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s(-1)) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

Alkyl and Aryl Transferases↗
PubMed1989

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells.

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

Amino Acid Sequence↗
PubMed1986

The accumulation of pyrophosphate by rat hepatocytes.

Hepatocytes that were isolated from 48 hr starved rats and incubated in Krebs-Henseleit bicarbonate buffer containing 10 microM A23187, 10mM l-lactate, 1mM pyruvate and 2mM l-lysine were found to contain 0.064 mumol of inorganic pyrophosphate/g wet wgt cells. Addition of either 20mM acetate or butyrate, which caused the formation of pyrophosphate in both the cytosol and the mitochondrial matrix or only the matrix, resulted in an increase of 0.915 and 1.91 mumol pyrophosphate/g wet wgt cells, respectively. The accumulation of pyrophosphate was shown to be non-linear with time and dependent on the calcium concentration of the incubation media. In contrast, incubations containing a combination of 10 mM NH4Cl and 5 mM ornithine, which resulted in the formation of pyrophosphate only in the cytosol, had a pyrophosphate content of 0.032 mumol/g wgt cells. When isolated hepatocytes that had been incubated with acetate or butyrate were subjected to digitonin fractionation, all of the recoverable pyrophosphate was present in the particulate fraction. It is concluded that pyrophosphate accumulates in isolated rat hepatocytes only in the presence of calcium and a calcium ionophore, only within the mitochondrial matrix and only when pyrophosphate is formed within the mitochondrial matrix.

Animals↗
PubMed1977

Evaluation of myocardial uptake of 99mtechnetium pyrophosphate in clinical exercise-induced ventricular ischemia.

Although acute infarction of the myocardium is known to accumulate 99mtechnetium pyrophosphate, it is not entirely clear that ischemia alone without necrosis does not result in abnormal uptake of 99mtechnetium pyrophosphate. The present study investigates whether transient myocardial ischemia is associated with localization of 99mtechnetium pyrophosphate by evaluating images obtained with the scintillation camera at rest and after exercise in 15 patients with unequivocal myocardial ischemia. All patients had angina pectoris, multivessel coronary artery stenoses by selective arteriographic studies, and electrocardiographic ischemic responses on treadmill exercise. Eleven of the 15 patients also underwent radionuclide imaging with 81rubidium at rest and after exercise; the results demonstrated scintigraphic ischemia. The scintiscans with 99mtechnetium pyrophosphate revealed no evidence of increased myocardial radioactivity after exercise compared to rest in 14 of the 15 patients. In contrast, myocardial activity was observed with 99mtechnetium pyrophosphate after treadmill exertion in the remaining patient, in whom a small subendocardial infarction appeared to have occurred with the exercise. It is concluded from these results that transient myocardial ischemia does not cause localization of 99mtechnetium pyrophosphate. These findings support the specificity of abnormal localization of 99mtechnetium pyrophosphate for acute myocardial infarction.

Adult↗
PubMed1990

Comparison of the anticalculus effect of two soluble pyrophosphate dentifrices with and without a copolymer.

A two phase, six month, double blind clinical study was conducted to compare the effect on supragingival calculus deposits of a dentifrice containing 1.30% soluble pyrophosphate (from 2.0% tetrasodium pyrophosphate) with and without the presence of 1.50% of a copolymer of methoxyethylene and maleic acid. In Phase I of the study, male and female adult subjects were stratified into two balanced groups according to baseline calculus scores. They received an oral prophylaxis and were assigned to the use of either a dentifrice containing 1.30% soluble pyrophosphate and 1.50% copolymer or to a placebo dentifrice that did not contain the anticalculus ingredients. After three months use of the products, the subjects received a calculus examination. The subjects were then entered into Phase II of the study where they were restratified into two balanced groups. They again received an oral prophylaxis and were assigned to the use of either a dentifrice containing 1.30% soluble pyrophosphate with no copolymer or to a placebo dentifrice. The results from the three month calculus examination indicated that the dentifrice containing soluble pyrophosphate and a copolymer reduced supragingival calculus deposits by 29.54%, as compared to the placebo dentifrice (less than 99% level of confidence). The results from the six month calculus examinations indicated that the dentifrice containing soluble pyrophosphate without the copolymer did not provide a statistically significant reduction in supragingival calculus after an oral prophylaxis. Thus, it is concluded that the presence of the copolymer in the soluble pyrophosphate dentifrice was essential for obtaining a statistically significant anticalculus effect.

Adult↗
PubMed1989

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene.

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.

Bicyclic Monoterpenes↗
PubMed1986

On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms.

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.

Bacteriophage phi X 174↗
PubMed1988

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (-)-endo-fenchol.

The conversion of geranyl pyrophosphate to (-)-endo-fenchol is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with a preparation of (-)-endo-fenchol cyclase (synthase) from common fennel (Foeniculum vulgare) gave labeled product of unchanged 3H:14C ratio in both cases, and each was dehydrated to a mixture of alpha- and beta-fenchene which were oxidized to the corresponding alpha- and beta-fenchocamphorones, again without change in isotope ratio. The location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The findings indicated that the configuration at C1 of the substrate was retained in the enzymatic transformation to (-)-endo-fenchol which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to (3R)-linalyl pyrophosphate and cyclization of the latter via the anti-endo-conformer. These absolute stereochemical elements of the reaction sequence were confirmed by the enzymatic conversion of (3R)-1Z-[1-3H]linalyl pyrophosphate to (-)-endo-fenchol and by the location of the tritium in the derived fenchocamphorones as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to (-)-endo-fenchol.

Camphanes↗
PubMed1999

Transduction mechanisms of porcine chondrocyte inorganic pyrophosphate elaboration.

OBJECTIVE: To investigate cellular signaling mechanisms that influence chondrocyte production of inorganic pyrophosphate (PPi), which promotes calcium pyrophosphate dihydrate (CPPD) crystal deposition. METHODS: Articular chondrocyte and cartilage cultures were stimulated with protein kinase C (PKC) activator and adenyl cyclase activator. Generation of extracellular PPi was measured. RESULTS: Adenyl cyclase activation resulted in diminished pyrophosphate generation. PKC activation stimulated pyrophosphate elaboration. CONCLUSION: Two signaling pathways, cAMP and PKC, modulate generation of extracellular pyrophosphate by cartilage and chondrocytes. They are novel targets for potentially diminishing extracellular pyrophosphate elaboration that leads to CPPD crystal deposition.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2006

Titanium implant osseointegration with calcium pyrophosphate in rabbits.

The objective of this study was to characterize calcium pyrophosphate material, evaluate its in vitro cytotoxicity, and assess its ability to induce bone formation. X-ray diffraction (XRD) was used to determine crystallinity and phases present in material. Serial dilutions of extracts, from 10-day dissolution tests in modified Eagle's medium, were exposed for 24 h to mouse fibroblasts and cytotoxicity assessed via viable staining. In vivo performance was determined by placing Ti screws with and without calcium pyrophosphate agglutinated with marrow adipose tissue in the tibiae of eight rabbits. New bone formation around test and control implants was evaluated histomorphometrically by using three fluorochrome labels: alizarin, calcein, and tetracycline. After 8 postoperative weeks, the animals were killed and specimens were retrieved and processed for fluorescence and light microscopic analysis. Calcium pyrophosphate showed no cytotoxicity and the XRD showed that the main phase of the analyzed sample corresponded to beta-calcium pyrophosphate. The largest fluorochrome labeling area occurred during the fourth and fifth postoperative weeks, in both control and experimental groups. Histologically, the bone neoformation occurred in regions where the calcium pyrophosphate was resorbed. The morphometric analysis showed implants placed with calcium pyrophosphate resulted in smaller polyfluorochrome labeling area (p < 0.05).

Animals↗
PubMed1983

Mechanistic aspects of the interaction of 99mTc in association with stannous pyrophosphate with damaged red blood cells.

The binding of the various components of [99mTc] stannous pyrophosphate and stannous pyrophosphate/pertechnetate to damaged red blood cells is studied. It is shown that the pyrophosphate molecule enters the damaged red blood cells when the pyrophosphate concentration in blood is greater than 100 nmol/ml but does so as the uncomplexed ion. Uptake of 99mTc when introduced as [99mTc]stannous pyrophosphate is constant at approximately 18%. If the 99mTc is introduced as pertechnetate after the damaged cells are mixed with stannous pyrophosphate then at low stannous ion concentrations the uptake is directly dependent on the stannous ion concentration. However, at higher stannous ion concentrations the uptake of technetium by the damaged red cells decreases, but this decrease appears to result from several independent aspects of the sample, such as the binding of the technetium to the plasma proteins and the displacement of the technetium by pyrophosphate within the damaged cell.

Erythrocytes↗
PubMed1984

Mean ionic charge on two components of technetium pyrophosphate prepared using stannous chloride.

Ionic charge, complexation equilibria, and acid-base equilibria can be measured at tracer concentrations by ion-exchange chromatography. To characterize better the principal components found in [99mTc]pyrophosphate preparations used for diagnostic bone and heart scanning, these were studied in two ion-exchange chromatographic systems: (A) DEAE-cellulose in pyrophosphate form at pH 6.0-7.5, and (B) DEAE-cellulose in perchlorate form at pH 3.7-4.8. The chromatographic retention times were measured as a function of pH and electrolyte concentration, and compared with those of trisoxalatochromium(III) complex, which was chosen as reference ion because of its similarity in charge and retention to the Tc-pyrophosphate complexes. The pyrophosphate complexes were even more sensitive to electrolyte concentration than was the triply negative reference ion: the calculated mean net charge in the mobile phase for the two principal Tc-pyrophosphate species were -4.5 +/- 0.5 and -4.9 +/- 0.5 at pH 4.3, and -11.2 +/- 1.3 and -10.1 +/- 1.0 at pH 7.0. It can be concluded that the two principal radioactive components in clinical Tc-pyrophosphate preparations both bear a high, pH-dependent negative charge.

Chromatography, Ion Exchange↗
PubMed2006

Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.

Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

Crystallography, X-Ray↗