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At least 793 records · Page 44Linked to original sources
PubMed1997

Homology modeling of Neurospora crassa geranylgeranyl pyrophosphate synthase: structural interpretation of mutant phenotypes.

A model of the tertiary structure of the Neurospora crassa carotenogenic prenyltransferase, geranylgeranyl pyrophosphate synthase (GGPPS), is presented, based on structural homology with other prenyltransferases and on the crystal structure of recombinant avian farnesyl pyrophosphate synthase (FPPS). The conserved aspartate-rich motifs DDxx(xx)D and associated basic residues, considered to be the active sites for binding and catalysis in all prenyltransferases, are highly conserved in the N. crassa GGPPS protein, while other regions display a lower degree of sequence homology; thus the GGPPS model structure is predicted to be highly reliable in the active site region. A number of carotene-deficient mutants have been generated utilizing the repeat-induced point mutation (RIP) mechanism: mutant al-3RIP1 carries a Ser-to-Asn mutation in position 336 which falls within the predicted active site of the enzyme. Analysis of the model structure of this mutant indicates that Ser336 may be involved in substrate uptake. Two other mutants, al-3RIP3 and al-3RIP6, carry mutations in positions in the GGPPS protein, homologous to regions of the avian FPPS enzyme proposed to be involved in enzyme dimerization and substrate uptake, respectively, suggesting an explanation for the reduced carotene content of these mutants.

Alkyl and Aryl Transferases↗
PubMed1991

Inorganic pyrophosphate, nucleoside triphosphate pyrophosphatase, and cartilage fragments in normal human synovial fluid.

The association between calcium pyrophosphate dihydrate (CPPD) crystal deposition and ageing is unexplained. Levels of inorganic pyrophosphate (PPi) and activity of nucleoside triphosphate pyrophosphatase (NTPP) were estimated in synovial fluid from 91 asymptomatic normal knees, including five with isolated CPPD crystal deposition (70 subjects; median age 47, range 22-83 years). In non-crystal fluids, PPi levels and NTPP activities were low (median, interquartile range: 9.5, 7.6-12.3 microM, and 1, 0.4-2 microM/30 min/mg protein, respectively: n = 50) and a generalized increase with age was not demonstrated. Higher values for PPi and NTPP in all five subjects with isolated CPPD deposition supports involvement of altered PPi metabolism, unrelated to ageing per se, in crystal formation. Presumed cartilage fragments were identified by morphological characteristics in 70% of normal fluids: metachromatic staining and electron microscopy, undertaken in a limited number, supported identification of such particles as cartilage. This finding supports dynamic turnover of healthy matrix, and questions the usefulness of identification, without quantification, cartilage fragments as a measure of cartilage damage.

Adult↗
PubMed1975

Pyrophosphate and diphosphonates in skeletal metabolism. Physiological, clinical and therapeutic aspects.

Pyrophosphate and diphosphonates produce striking results on calcium metabolism in experimental animals and man. Compounds containing P-O-P- bonds (e.g. inorganic pyrophosphate [PP-ii1 or P-C-P bonds (diphosponates) inhibit both the formation and dissolution of calcium phosphate crystals in vitro. PP-i may have a physiological function in regulating calcification and bone turnover, and obnormalities in its metabolism may occur in some human diseases notably hypophosphatasia and pseudogout. Diphosphonates inhibit ectopic calcification, and slow down resorption and bone turnover in several experimental systems in vivo. They have helped in studies of various aspects of the regulation of calcium metabolism. The diphosphonate, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) has been shown in clinical studies to be effective against ectopic calcification particularly in myositis ossificans progressiva and in disorders of increased bone resorption such as Paget's diseases and some types of osteoporosis. -99mTechnetium complexes of EHDP, PP-i and other polyphosphates have also recently been used successfully as bone scanning agents.

Animals↗
PubMed2002

Bilateral symptomatic synovial cysts of the lumbar spine caused by calcium pyrophosphate deposition disease: a case report.

STUDY DESIGN: A case of bilateral symptomatic facet joint synovial cysts arising in association with calcium pyrophosphate deposition disease is reported. OBJECTIVE: To present a previously unreported cause for symptomatic synovial cysts of the lumbar spine. SUMMARY OF BACKGROUND DATA: Synovial cysts of the facet joints occur most commonly in association with degenerative disease of the spine in older individuals. The association of these cysts with trauma, rheumatoid arthritis, spondylolysis, and kissing spinous processes also has been reported. These cysts can cause symptoms and signs from direct compression of the dura. Chondrocalcinosis has not been previously reported to cause symptomatic synovial cysts. METHODS: A 67-year-old woman presented with right lower limb sciatica caused by a right L4-L5 facet joint cyst, which resolved after surgical decompression. A year later, she presented with left lower limb sciatica caused by development of a new L4-L5 facet joint cyst, which also resolved after surgical decompression. RESULTS: Histopathologic examination of each cyst showed a cyst wall of fibrous tissue with synovial lining, inflammation, and granulation tissue. Examination of the tissue under polarized light showed positively birefringent, short blunt crystals of calcium pyrophosphate dihydrate. CONCLUSIONS: In patients with a history of gout or pseudogout, a rare possibility of a synovial cyst should be considered in the differential diagnosis during investigation for the cause of neural compression resulting in sciatic syndrome.

Aged↗
PubMed2000

Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing.

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

Alleles↗
PubMed1993

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber.

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).

Journal Article↗
PubMed1971

Assay of adenosine 5-triphosphate sulfurylase by pyrophosphate exchange.

The sulfate-dependent pyrophosphate exchange reaction has been re-examined and confirmed. Standard assay conditions for measuring ATP sulfurylase by sulfate-dependent pyrophosphate exchange are described and some properties of the enzyme (measured in crude dialyzed extracts) are reported. This method has many advantages over the well established molybdate method.

Journal Article↗
PubMed1976

Induction of Furano-terpene Production and Formation of the Enzyme System from Mevalonate to Isopentenyl Pyrophosphate in Sweet Potato Root Tissue Injured by Ceratocystis fimbriata and by Toxic Chemicals.

When sweet potato (Ipomoea batatas) root tissue was infected by Ceratocystis fimbriata, activity of the enzyme system from mevalonate to isopentenyl pyrophosphate, especially of pyrophosphomevalonate decarboxylase (EC 4.1.1.33), was increased in the noninfected tissue adjacent to the infected region, preceding the furano-terpene production in the infected region. Cutting and incubation of sweet potato slices did not produce furano-terpenes, and only slightly increased the activity of the enzyme system from mevalonate to isopentenyl pyrophosphate. The enzymic activity in diseased tissue was localized in the soluble fraction, and was higher in the tissue from the surface to a depth of about 5 mm with gradual decrease toward the inner part.Mercuric chloride (0.1%, w/v) and sodium dodecyl sulfate (1.0%, w/v) were utilized as model inducers of furano-terpenes and pyrophosphomevalonate decarboxylase. The mercuric chloride- or sodium dodecyl sulfate-induced response was inhibited by administration of cycloheximide to the discs together with the inducer immediately after disc preparation. When cycloheximide or blasticidin S was applied together with the inducer, to the discs 9 hours or more after disc preparation, the induction was not inhibited but rather stimulated.

Journal Article↗
PubMed1987

Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds.

THE DISTRIBUTION OF PYROPHOSPHATE: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles.min.milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K(m) values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P(2). The activation is caused by lowering the K(m) for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P(2) and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K(m) of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K(m) for fructose 6-P. The ATP content appears to be at saturating levels for PFK.

Journal Article↗
PubMed1989

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains.

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg(2+) and exhibited hyperbolic kinetics with all substrates including Mg(2+). K(m) values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. Kinetic constants were determined in the presence of fructose-2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K(i) values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.

Journal Article↗
PubMed1991

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje.

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2 degrees C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4 degrees C. During storage at 8 degrees C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8 degrees C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8 degrees C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4 degrees C but remained the same throughout storage at 8 degrees C. After exposure to 2 degrees C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4 degrees C but decreased slightly at 8 degrees C. All observed changes became annulled after transfer of cold stored tubers to 18 degrees C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.

Journal Article↗
PubMed1991

Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits.

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (alpha-subunit) and 65 (beta-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in beta-subunit and twofold increase in alpha-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total beta-subunit to alpha-subunit content. The beta- to alpha-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.

Journal Article↗
PubMed2003

KHCoP2O7.2H2O: a novel acidic pyrophosphate.

Potassium cobalt hydrogenpyrophosphate dihydrate, KHCoP(2)O(7).2H(2)O, crystallizes in the orthorhombic space group Pnma. This salt is isotypic with KHMP(2)O(7).2H(2)O (M = Mn and Zn). The structure consists of alternating layers, built from HP(2)O(7)(3-) acidic pyrophosphate groups and CoO(6) octahedra, joined by potassium ions and bridging hydrogen bonds. The Co, K and water O atoms lie on mirror planes. The pyrophosphate group consists of two symmetry-related PO(4) groups, with the bridging O atom on a mirror plane.

Journal Article↗
PubMed1990

Structures of the meridional and facial isomers of triamminechromium pyrophosphate dihydrate.

The meridional and facial isomers of triamminechromium pyrophosphate were separated by chromatography on Dowex-50-H+, and crystallized as isoionic species. The meridional isomer crystallized as a monomer [Cr(HP2O7)(NH3)3(H2O)].2H2O but the facial isomer crystallized as a centrosymmetric dimer of [Cr(HP2O7)(NH3)3]. 2H2O, in which the remaining water in the coordination sphere of each chromium is replaced by a phosphate oxygen from the other monomer unit. Meridional isomer: monoaquatriammine(pyrophosphato)chromium(III) dihydrate, Mr = 332.1, monoclinic, P21/c, a = 7.825 (2), b = 10.107 (3), c = 15.322 (5) A, beta = 103.92 (5) degrees, V = 1176 (1) A 3, Z = 4, Dx = 1.875 g cm-3 lambda(Mo K alpha) = 0.71073 A, mu = 12.6 cm-1, F(000) = 684, final R = 0.050 for 1828 reflections. The most notable difference between this compound and the corresponding tetraammine complex reported previously is a significant shortening of the metal-ligand bond lengths not only for the water ligand but also for the ammonia ligands. The dimer: mu-(pyrophosphato-O,O',O")-bis[triamminechromium(III)] dihydrate, Mr = 314.0, monoclinic, P21/c, a = 8.695 (2), b = 10.327 (3), c = 11.913 (4) A, beta = 97.81 (5) degrees, V = 1060 (1) A3, Z = 4, Dx = 1.969 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 125.7 cm-1, F(000) = 644, final R = 0.047 for 1389 reflections. This structure, which sits on a center of inversion, forms a tricyclic complex involving two Cr atoms. The structure is characterized by reciprocal coordination between the metal ions and anionic O atoms of the pyrophosphate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Structure↗
PubMed2006

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human phosphoribosyl pyrophosphate synthetase 1 (PRS1).

Phosphoribosyl pyrophosphate synthetase (PRS; EC 2.7.6.1) catalyzes the reaction of ribose-5-phosphate (R5P) with ATP to yield AMP and PRPP (5-phosphoribosyl-1-pyrophosphate), which is necessary for the de novo and salvage pathways of purine-, pyrimidine- and pyridine-nucleotide biosynthesis. PRPP is a metabolite that is required at all times in the cell and is thus central to life. In this study, human PRS1 was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals in complex with Mg2+, inorganic phosphate (P(i)) and ATP were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.6 A resolution. The crystal belongs to space group R3, with unit-cell parameters a = b = 168.846, c = 61.857 angstroms, assuming two molecules in the asymmetric unit and a volume-to-weight ratio of 2.4 angstroms3 Da(-1), which was consistent with the result calculated from the self-rotation function.

Brain↗
PubMed1995

Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A.

Lipopolysaccharides (LPS) of two polymyxin-resistant (pmr) mutants and the corresponding parent strain of Escherichia coli were chemically analysed for composition and subjected to 31P-NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild-type and pmr LPS, the latter contained two- to threefold higher amounts of 2-aminoethanol. The pmr LPS also contained 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4'-phosphate of lipid A. Comparative 31P-NMR analysis of the de-O-acylated LPS of the wild-type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71-79%) diphosphate diesters, which accounted for only 20% in the wild-type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild-type LPS. In the lipid A of the pmr strains, the 4'-phosphate was to a significant degree (35%) substituted by L-Arap4N, whereas in the wild-type LPS the L-ArapN was absent. In the pmr lipid A, 2-aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild-type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4'-monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2-aminoethanol.

Amino Sugars↗
PubMed1982

Hereditary pyrophosphate arthropathy (familial articular chondrocalcinosis) in Sweden.

Genealogical links between three Swedish families with hereditary pyrophosphate arthropathy were found in the 18th century, indicating a possible founder effect, similar to earlier findings in Slovakia, France and Chile. However, no connection between the Swedish and other European families with the disease has so far been found. In accordance with other reported familial aggregations of pyrophosphate arthropathy, the transmission of the disease in the Swedish families appeared to be autosomal dominant with incomplete penetrance and variable expressivity. Severe symptoms related to homozygotic cases reported in some other families were not found in Sweden.

Adult↗