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PubMed1985

99mTc bone scanning agents--I. Influence of experimental conditions on the formation and gel chromatography of 99mTc(Sn)pyrophosphate complexes.

The conversion of 99mTcO4- to 99mTc(Sn)pyrophosphate complexes was investigated under various experimental conditions. An increase of the Sn(II) concentration had a beneficial effect, whereas the ligand concentration had little effect. The pH had only a small influence over the range 2-8. Raising the pH to 10 resulted in the partial decomposition of the complexes, which could be reversed by lowering the pH. Furthermore, the occurrence of various complexes was investigated by means of gel chromatography on Biogel P-4 as a function of pH and of the Sn(II) and pyrophosphate concentrations. Four major fractions were found. A single preparation contained, however, no more than two major fractions. The formation of the different complexes was mainly governed by the pH and the ligand concentration. The influence of the eluent on the decomposition and interconversion of the complexes during chromatography was also studied. It appeared to be necessary that the eluent should have the same composition (except for 99mTcO4-) as the reaction mixture.

Bone and Bones↗

PubMed1981

The phosphogluconate pathway and synthesis of 5-phosphoribosyl-1-pyrophosphate in human fibroblasts.

The phosphogluconate pathway (pentose phosphate cycle) of normal fibroblasts was stimulated 20-fold by methylene blue and inhibited to 14% of the baseline rate by 6-aminonicotinamide. In fibroblasts deficient in glucose-6-phosphate dehydrogenase activity (an average of 1.5% of normal mean), the pentose phosphate cycle was unaffected by either methylene blue or 6-aminonicotinamide. In normal cells, neither the intracellular concentration nor the rate of generation of 5-phosphoribosyl-1-pyrophosphate was altered by the marked and opposite changes in the rate of the phosphogluconate pathway caused by methylene blue and 6-aminonicotinamide. Intracellular ribose 5-phosphate concentration was increase by methylene blue (an average increase of 83%) but not significantly altered by 6-aminonicotinamide. In fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the 5-phosphoribosyl-1-pyrophosphate concentration and rate of generation were higher rather than lower in comparison to normal cells under all conditions studied. The data suggest a predominant role for the nonoxidative branch of the phosphogluconate pathway in supplying ribose 5-phosphate for nucleotide biosynthesis. Pentose phosphate supply cannot be considered an essential function of the oxidative branch in fibroblasts.

Cell Line↗

PubMed1988

99mTc bone scanning agents--IV. Chemical characterization of 99mTc(Sn)-pyrophosphate complexes.

Various reaction mixtures for the preparation of 99Tc(Sn)-pyrophosphate were investigated by means of gel chromatography. All components were radioactively labeled. The most likely composition of the complexes, which also appear in "no carrier added" preparations, was determined. At pH 7 one complex is found with the composition TcPyp2. Two complexes are found at pH 4: TcPyp and TcPyp2. Further, at pH 7 a polymeric technetium compound is found not containing tin or pyrophosphate.

Bone and Bones↗

PubMed2005

Bioactive calcium pyrophosphate glasses and glass-ceramics.

Calcium phosphate glass-based materials in the pyrophosphate region are briefly reviewed. Calcium pyrophosphate glasses can be prepared by including a small amount of TiO(2) (<or=10mol%). Bonelike apatite forms on some of the glasses in simulated body fluid. By heating powder-compacts of the glasses, they are crystallized and subsequently are sintered, resulting in fabrication of high-strength glass-ceramics with machinability; they are easier to be machined using conventional tools in comparison with conventional calcium phosphate ceramics. beta-Ca(2)P(2)O(7) crystal formed in the glass-ceramics plays an important role in the machinability. Their apatite-forming ability in simulated body fluid is drastically enhanced after autoclaving in distilled water. The glass-ceramics can be easily coated on a new beta-type titanium alloy using a conventional glazing technique.

Alloys↗

PubMed2003

An efficient enzymatic synthesis of thiamin pyrophosphate.

Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.

Base Sequence↗

PubMed2005

Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.

Anti-Infective Agents↗

PubMed1984

Measurement of myocardial infarct size by technetium pyrophosphate single-photon tomography.

The primary determinant of prognosis after acute myocardial infarction (AMI) is the size of the acute infarct. The present study evaluates 46 patients with different infarct distributions and sizes to test the hypothesis that single photon emission computed tomography with technetium-99m pyrophosphate (Tc-99m-PPi) and blood pool overlay allows measurements of AMI size that provide insight into prognosis irrespective of infarct location. Identical Tc-99m-PPi and ungated blood pool projections were acquired over 180 degrees with a rotating gamma camera. Reconstructed sections were color-coded and superimposed for purposes of infarct localization. Areas of increased pyrophosphate uptake within myocardial infarcts were thresholded at 65% of peak activity. The blood pool was thresholded at 50% and subtracted so as to determine an endocardial border for the left ventricle. Using this method, myocardial infarcts weighed 2.5 to 81.2 g. The correlation of infarct mass with prognosis showed that patients without previous AMI and with acute infarcts that weighed more than 40 g had an increased frequency of death and congestive heart failure (p less than 0.001). The correlation of measured infarct mass with peak serum creatine kinase level was significant (r = 0.83, p less than 0.001; y = 0.015x + 13.20). The correlation coefficients for anterior, inferior and nontransmural AMI were not significantly different from those for the entire group. In conclusion, tomographically determined infarct mass data correlate with subsequent clinical prognosis, and Tc-99m-PPi tomography with blood pool overlay is a safe and effective means of sizing infarcts in patients with AMI.

Adult↗

PubMed2000

The formation of a 1-5 phosphodiester linkage in the spontaneous breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate.

The decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) in the presence of Mg2+ at pH=7.8 yields a combination of products including ribose 5-phosphate, ribose 1-phosphate, 5-phosphoribosyl 1,2 cyclic phosphate, inorganic phosphate, and pyrophosphate. Hydrogen decoupled 31P NMR analysis of the product mixture also exhibits a sharp peak (+2.6 ppm from phosphocreatine) in a chemical shift region which includes phosphodiester bonds. Alkaline phosphatase treatment of the product mixture results in cleavage of monophosphate esters such as ribose 1-phosphate and ribose 5-phosphate, but does not affect the unidentified peak. Homonuclear (1H) correlation spectroscopy (COSY) of a partially purified sample was successful in identifying the hydrogen spectra of this compound. Combined with results from the splitting patterns of selectively decoupled 31P spectra, the COSY data indicate that several hydrogens are directly coupled to the unknown phosphate group with J value matches to the hydrogen on carbon one and to the two hydrogens on carbon five. Heteronuclear (1H-31P) chemical shift correlation studies confirm these couplings and further substantiate the formation of a ribose 1-5 phosphate linkage during the degradation of PRPP under these conditions. It is presently unknown whether this is an intramolecular or intermolecular phosphodiester linkage, although some spectroscopic evidence suggest the intramolecular bond formation, i.e. a ribose 1,5-cyclic phosphate (R-1,5cP). The formation of R-1,5cP helps explain the observation that the 5-phosphate group from PRPP becomes labile during the spontaneous degradation of PRPP.

Diphosphates↗

PubMed2003

The catalytic effect of Mg2+ and imidazole on the decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate in aqueous solution.

The rate of decomposition of aqueous solutions of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) is greatly enhanced by the presence of certain divalent ions. High levels of Mg(2+) were shown to increase the rate of phosphate removal from PRPP by factors greater than 100-fold. The combined action of Mg(2+) and a nitrogenous compound (imidazole was employed in this study) increases the degradation rate constants by up to 1000-fold. The degradation of PRPP in neutral solutions follows two main paths, a pyrophosphate hydrolysis reaction yielding ribose 5-phosphate and an internal beta-phosphate cleavage yielding 5-phosphoribosyl-1,2 cyclic phosphate. The catalytic effect of Mg(2+) on PRPP degradation appears to suggest a connection between the observed rate enhancements and the dimagnesium form of PRPP. Rate enhancing effects of Mg(2+) are greatest at pH values (i.e. pH>7) which promote strong phosphate-Mg(2+) interaction.

Catalysis↗

PubMed1983

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.

Borohydrides↗

PubMed2006

EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.

Electron Spin Resonance Spectroscopy↗

PubMed2002

[Calcium pyrophosphate deposits--a chameleon].

Calcium Pyrophosphate Dihydrate (CPPD) crystals deposit in articular fibro- or hyaline cartilage (chondrocalcinosis), joint capsules, synovium, periarticular ligaments and tendons resulting in an age dependent prevalence. These calcifications may be asymptomatic or may manifest as acute pseudogout arthritis, pseudorheumatoid arthritis, bursitis, tenosynovitis, tendinitis, polymyalgic syndrome or chronic pyrophosphate arthropathy. The diagnosis is based on the presence of intracellular CPPD crystals in synovial fluid detected by polarizing microscopy, the characteristic radiological changes and the typical clinical presentations. The therapy is symptom oriented or disease specific in case of an underlying metabolic disease such as hemochromatosis, hyperparathyroidism, hypophosphatasia, hypomagnesemia or hypothyroidism.

Calcium Pyrophosphate↗

PubMed1999

Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis.

Guanine phosphoribosyltransferase (GPRTase) from Giardia lamblia, an enzyme required for guanine salvage and necessary for the survival of this parasitic protozoan, has been kinetically characterized. Phosphoribosyltransfer proceeds through an ordered sequential mechanism common to many related purine phosphoribosyltransferases (PRTases) with alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) binding to the enzyme first and guanosine monophosphate (GMP) dissociating last. The enzyme is a highly unique purine PRTase, recognizing only guanine as its purine substrate (K(m) = 16.4 microM) but not hypoxanthine (K(m) > 200 microM) nor xanthine (no reaction). It also catalyzes both the forward (kcat = 76.7 s-1) and reverse (kcat = 5.8.s-1) reactions at significantly higher rates than all the other purine PRTases described to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high K(m) for pyrophosphate (PPi) (323.9 microM) in the reverse reaction, comparable only with the high K(m) for PPi (165.5 microM) in Tritrichomonas foetus HGXPRTase-catalyzed reverse reaction. As the latter case was due to the substitution of threonine for a highly conserved lysine residue in the PPi-binding loop [Munagala et al. (1998) Biochemistry 37, 4045-4051], we identified a corresponding threonine residue in G. lamblia GPRTase at position 70 by sequence alignment, and then generated a T70K mutant of the enzyme. The mutant displays a 6.7-fold lower K(m) for PPi with a twofold increase in the K(m) for PRPP. Further attempts to improve PPi binding led to the construction of a T70K/A72G double mutant, which displays an even lower K(m) of 7.9 microM for PPi. However, mutations of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPRTase, suggesting the requirement of flexibility in the putative PPi-binding loop for enzyme catalysis, which is apparently maintained by the glycine residue. We have thus tentatively identified the PPi-binding loop in G. lamblia GPRTase, and attributed the relatively higher catalytic efficiency in the forward reaction to the unusual loop structure for poor PPi binding in the reverse reaction.

Animals↗

PubMed1999

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids.

In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.

Amino Acid Sequence↗

PubMed1996

Inhibition of calcium pyrophosphate dihydrate crystal formation in articular cartilage vesicles and cartilage by phosphocitrate.

Articular cartilage vesicles (ACV), isolated by differential centrifugation of adult hyaline articular cartilage collagenase digests, mineralized in the presence of calcium and ATP. Mineral analysis by microscopy, chemical analysis, energy-dispersive analysis, and infrared spectroscopy revealed crystals resembling calcium pyrophosphate dihydrate (CPPD). Adult articular cartilage also underwent ATP-dependent mineralization, supporting the contention that vesicles in situ fostered adult articular cartilage mineralization. Phosphocitrate (PC) is a recognized in vitro inhibitor of hydroxyapatite and calcium oxalate monohydrate crystal formation, but it is not known whether PC can similarly restrict CPPD crystal development. In the present study we examine the effect of PC, citrate, and n-sulfo-2-amino-tricarballylate (SAT, a PC analogue) on the ATP-induced CPPD crystal formation in both ACV and articular cartilage models. Only PC (10-1000 microM) blocked both the ATP-dependent and -independent mineralization in ACV in a dose-dependent fashion. At 1 mM, SAT and citrate blocked the ATP-independent mineralization. Similarly, only PC blocked both the ATP- and non-ATP-dependent mineralization in native articular cartilage slices. PC, SAT, and citrate had no effect on ACV nucleoside triphosphate pyrophosphohydrolase activity, suggesting that none of these agents blocked mineralization through the inhibition of nucleoside triphosphate pyrophosphohydrolase activity, which generates inorganic pyrophosphate from ATP.

Adenosine Triphosphate↗

PubMed2000

Density functional geometry optimization and energy calculations of calcium(II)-triphosphate complexes. Polyphosphates as possible dissolving agents for calcium pyrophosphate dihydrate crystals in chondrocalcinosis disease.

Geometry optimizations and energy calculations have been carried out via molecular orbital methods at the density functional B3LYP/LANL2DZ level on the molecules PO3-, OPO3(3-), HOPO3(2-), CH3OPO3(2-), H(CH3OPO3)-, O(PO3)2(4-), HO(PO3)2(3-), CH2(PO3)2(4-), (CH3OPO2)O(PO3)3-, O(PO3)3(5-), HO(PO3)3(4-), (PO3)3(3-), (CH3OPO2)O(PO3)2(4-), [Mg[O(PO3)2)]]2-, [Ca[O(PO3)2]]2-, [Ca[CH2(PO3)2]]2-, [Ca[CH3OPO2)O(PO3)]]-, [Ca(PO3)3]-, [Ca[O(PO3)3]]3-, and [Ca[CH3OPO2)O(PO3)2]]2- with the aim to find reliable and easily accessible computational methods to simulate some phosphate-containing molecules of importance for the living cells and to study the energetics for protonation and metal-complex formation reactions. The analysis is part of a general investigation on phosphate-containing molecules as potential dissolving agents for calcium pyrophosphate dihydrate (CPPD) crystals which deposit in certain articular diseases. The basis set was expanded to 6-31G** for the P atoms for all the molecules investigated and to 6-31G* for the O atoms for OPO3(3-). Calculations at the semiempirical MNDO/d level were also carried out for comparison purposes on the free ligand molecules and on [Mg[O(PO3)2]]2-. The density functional analysis reproduced well the geometry found at the solid state via X-ray diffraction. The analyses of the geometrical parameters and the total electronic energy of the molecules shows that O(PO3)2(4-) and other di- and tri-phosphates are versatile ligands for divalent metal ions like Ca2+. The computed P-O-P bond angle for free O(PO3)2(4-) is 180 degrees and the conformation of the two PO3- groupings is staggered along the P...P vector. The linear arrangement for P-O-P is assisted by P-O pi interactions. The bending of the P-O-P angle when accompanied by a slight P-O(b) elongation requires a very small amount of energy; 4.65 kcal/mol to pass from 180 to 140 degrees , as calculated at the DFT level. The computed Ca-O and Mg-O bond distances for [M[O(PO3)2]]2- are 2.378 and 2.079A, when the metal ions link two oxygen atoms from each PO3 group. The computed Ca-O bond lengths for [Ca[CH3OPO2)O(PO3)]]- are 2.482 (PalphaO2) and 2.358A (PbetaO2), showing a significant lengthening for Ca-OPalpha, when compared to the pyrophosphate derivative. The Ca-O bond lengths for [Ca[O(PO3)3]]3- and [Ca[CH3OPO2)O(PO3)2]]2- are 2.251A and 2.525 (PalphaO2), 2.407 and 2.338 (PbetaO2), and 2.251 and 2.228A (PgammaO2), showing a shortening for the Ca-OPgamma bond upon methylation. The (Pbeta)O-Pgamma bond length increases significantly (0.09 A) upon Ca(II) coordination to (CH3OPO2)O(PO3)2(4-) via all the three PO3 groups. This latter result suggests that metal complexes of linear organic-triphosphates have a larger tendency to release the PgammaO3 group when compared to the free ligand molecules. The electronic contribution to the energy of the complex formation reaction for [Ca[CH2(PO3)2]]2- is only slightly higher (some 1.8 kcal) than that for [Ca[O(PO3)2]]2-; but is much higher (some 63 kcal) than that relevant to the formation of [Ca[CH3OPO2)O(PO3)2]]2-. (ABSTRACT TRUNCATED)

Calcium Phosphates↗

PubMed1999

Refinement of the chromosome 5p locus for familial calcium pyrophosphate dihydrate deposition disease.

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.

Calcium Pyrophosphate↗

PubMed2002

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH.

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Amino Acid Sequence↗