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PubMed1981

Arginyl-tRNA synthetase from Baker's yeast. Order of substrate addition and action of ATP analogs in the aminoacylation reaction; influence of pyrophosphate on the catalytic mechanism.

The order of substrate addition to arginyl-tRNA synthetase from baker's yeast has been investigated by bisubstrate kinetics, product inhibition and inhibition by three different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3'-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates. The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the three substrates and release of the products in random order. The different inhibitors are bound to different enzyme . substrate complexes of the reaction sequence. Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.

Acylation↗
PubMed1986

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient.

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
PubMed1989

Inhibitory effect of pyrophosphate, citrate, magnesium and chondroitin sulphate in calcium oxalate urolithiasis.

The inhibitory capacity of pyrophosphate, citrate, magnesium and chondroitin sulphate was investigated, using the urine of 21 calcium oxalate stone-forming patients without metabolic alterations. The inhibitory effect of these substances was assessed by a combination of nephelometry (light scattering) and optical microscopy. The results showed that citrate and magnesium had an inhibitory effect in a significant number of cases. Pyrophosphate and chondroitin sulphate had a less marked effect. The main urinary lithogenic biochemical parameters of the patients were also studied to see if there was a relationship between them and the inhibitory capacity of the compounds.

Calcium Oxalate↗
PubMed1996

Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation.

Pyrophosphate can serve as a source of phosphoryl groups for the phosphorylation of E. coli proteins. The main target of such phosphorylation is a 49-kDa protein which is covalently modified at serine. The same phosphorylation pattern is obtained in the presence of ATP, which suggests that pyrophosphate can substitute for ATP for bacterial protein phosphorylation.

Autoradiography↗
PubMed2001

Biochemical characterization of an active pyrophosphate-dependent phosphofructokinase from Treponema pallidum.

An active pyrophosphate-dependent phosphofructokinase containing a six residue polyhistidine tag has been cloned from Treponema pallidum, and characterized biochemically. The phosphofructokinase has pH optima for activity of 8.0 for both the forward and reverse reactions. The apparent K(m) for pyrophosphate was 0.042 mM (V(max) of 141 U mg(-1) protein) and for fructose-6-phosphate, 0.529 mM. The apparent K(m) for the reverse reaction for fructose-1,6-diphosphate was 0.267 mM (V(max) of 42.4 U mg(-1) protein). The enzyme appears to be both a dimer and non-allosteric.

Binding, Competitive↗
PubMed1976

Histochemical localization of alkaline pyrophosphate- phosphohydrolase in tooth-forming cells of rat.

The activity of a pyrophosphate-splitting tissue factor in jaws, teeth and intestinal mucosa has been studied by means of histochemistry. Freeze-cut, unfixed sections of whole animals were incubated in a buffered medium (pH 8.6) containing inorganic pyrophosphate (PP) Pb2+, Mg2+, and Zn2+ at various concentrations. The effects of compound R 8231, heat, aldehyde fixation, and demineralization with EDTA were also investigated. In sections showing optimal staining, deposition of incubation products was found in the stratum intermedium and the subodontoblastic cells of the developing tooth, in the osteoblastic layers, and at the surface of the intestinal mucosa. The hard tissues were also stained except in the demineralized sections. Treatment with heat or compound R 8231 resulted in loss of originally observed soft tissue staining while short-time demineralization with EDTA enhanced the staining reaction. It is argued that a nonspecific deposition of the capturing ion, Pb2+, can hardly explain the observed soft tissue staining. The results point to the presence of a PP-splitting enzyme, and it is suggested that the enzyme exhibits features of an alkaline phosphatase with PP-phospholhydrolytic properties rather than of an inorganic pyrophosphatase.

Animals↗
PubMed1998

J-104,871, a novel farnesyltransferase inhibitor, blocks Ras farnesylation in vivo in a farnesyl pyrophosphate-competitive manner.

Farnesylation of the activated ras oncogene product by protein farnesyltransferase (FTase) is a critical step for its oncogenic function. Because squalene synthase and FTase recruit farnesyl pyrophosphate as a common substrate, we modified squalene synthase (SS) inhibitors to develop FTase inhibitors. Among the compounds tested, a novel FTase inhibitor termed J-104,871 inhibited rat brain FTase with an IC50 of 3.9 nM in the presence of 0.6 microM farnesyl pyrophosphate (FPP), whereas it scarcely inhibited rat brain protein geranylgeranyltransferase-I or SS. The in vitro inhibition of rat brain FTase by J-104,871 depends on the FPP concentration but not on the concentration of Ras peptide. Thus, in vitro studies strongly suggest that J-series compounds have an FPP-competitive nature. J-104,871 also inhibited Ras processing in activated H-ras-transformed NIH3T3 cells with an IC50 value of 3.1 microM. We tested the effects of lovastatin and zaragozic acid A, which modify cellular FPP levels, on Ras processing of J-104,871. Lovastatin, a hepatic hydroxymenthyl coenzyme A reductase inhibitor that reduced the cellular FPP pool, increased the activity of J-104,871, whereas 3 microM zaragozic acid A, an SS inhibitor that raised the FPP level, completely abrogated the activity of J-104,871 even at 100 microM. These results suggest that J-104,871 inhibits FTase in an FPP-competitive manner in whole cells as well as in the in vitro system. Furthermore, J-104,871 suppressed tumor growth in nude mice transplanted with activated H-ras-transformed NIH3T3 cells.

3T3 Cells↗
PubMed2004

Phosphorylation of proteins by inositol pyrophosphates.

The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast. Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.

Adenosine Triphosphate↗
PubMed2002

Mn(III) pyrophosphate as an efficient tool for studying the mode of action of isoniazid on the InhA protein of Mycobacterium tuberculosis.

The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.

Antitubercular Agents↗
PubMed1998

PRS5, the fifth member of the phosphoribosyl pyrophosphate synthetase gene family in Saccharomyces cerevisiae, is essential for cell viability in the absence of either PRS1 or PRS3.

In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Deltaprs5 strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S. cerevisiae.

Base Sequence↗
PubMed1999

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene.

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.

Alkyl and Aryl Transferases↗
PubMed1962

Inactivation of the T-even coliphages by pyrophosphate.

Van Vunakis, Helen (Brandeis University, Waltham, Mass.) and Roger M. Herriott. Inactivation of the T-even coliphages by pyrophosphate. J. Bacteriol. 83:590-596. 1962.-Pyrophosphate liberates the deoxyribonucleic acid from the T-even coliphages. The reaction is dependent on pH, temperature, time, and ionic environment. The reaction can be inhibited by magnesium, putrescine, spermidine, and neutral molecules above a certain molecular weight. Possible mechanisms of the reaction are discussed.

Bacteriophages↗
PubMed1984

Prevalence of periarticular calcifications in pyrophosphate arthropathy and their relation to nodal osteoarthrosis.

X-rays of the shoulder, hand, and knee joints from 30 patients with pyrophosphate arthropathy (PA) and 30 age and sex matched control subjects were examined for periarticular, dense, homogeneous calcifications considered to be apatite deposits. They were found in 30% of the patients with PA compared with 3.3% of the controls. In addition in the PA group the incidence of Heberden's nodes was significantly increased in cases with periarticular calcifications, implying that mixed (calcium pyrophosphate dihydrate + apatite) crystal deposition disease could be related to nodal osteoarthrosis.

Adult↗
PubMed1996

Association between synovial fluid levels of inorganic pyrophosphate and short term radiographic outcome of knee osteoarthritis.

OBJECTIVE: To test the hypothesis that high concentrations of extracellular inorganic pyrophosphate (PPi), which associate with increased cell synthesis and turnover in cartilage, may act as a marker for structural outcome in knee osteoarthritis (OA). METHOD: One hundred and thirty five consecutive patients referred to hospital with knee OA (59 men, 76 women; mean age 71 years, range 41-88) were followed prospectively for a median of 2.5 years (interquartile range 1.75-3.0). Synovial fluid (SF) aspirated at presentation (202 OA knees: 68 bilateral, 66 unilateral) was assessed for PPi content by radiometric assay. Knee radiographs at presentation and at final review were assessed for change in global (Kellgren) and individual features (narrowing, osteophyte, sclerosis, cyst, attrition) of OA. RESULTS: The median SF PPi level was 10.5 mumol (range 0.07-72.4). At baseline, high PPi was significantly associated with presence of calcium pyrophosphate crystals, chondrocalcinosis, and bone attrition. Radiographic change was observed in 164 knees. High PPi levels were negatively associated with change in Kellgren and Lawrence grade, further narrowing, and increase in osteophyte, but positively associated with development of attrition. In the 68 patients from whom bilateral data were obtained, there was correlation between right and left knees for PPi levels, all baseline radiographic scores, and changes in radiographic features. Multiple logistic regression analysis for PPi as a continuous variable (age, gender, and patient number included in model) showed a negative correlation with change in global Kellgren and Lawrence grade (odds ratio (OR) 0.97, 95% confidence interval (CI) 0.95 to 0.99) and a positive correlation with attrition (OR 1.04, 95% CI 1.02 to 1.07). CONCLUSION: High SF levels of PPi are associated with favourable radiographic outcome in terms of progressive change in Kellgren grade. Such elevated PPi levels, however, may inhibit new bone formation and remodelling in knee OA.

Adult↗
PubMed1981

Advantages of technetium pyrophosphate scintigraphy over plasma enzyme analysis in estimation of anterior myocardial infarct size.

Infarct size was estimated by cumulative creatine kinase MB isoenzyme (CKMB-r) release and by technetium 99m stannous pyrophosphate (TcPYP) scintigraphy in 27 patients with acute anterior myocardial infarction. In eight patients, scintigraphy showed a central area of reduced tracer uptake surrounded by a peripheral rim of increased TcPYP accumulation ("doughnut" pattern). This appearance occurred only in large infarcts and the maximal scintigraphic area (51.3 +/- 2.8 cm2, mean +/- SEM) in this group was significantly greater than that in the remainder (28.1 +/- 2.5 cm2). Correlation between CKMB-r and maximal scintigraphic infarct area was moderate in the whole group. Exclusion of patients, however, with "doughnut" scintigrams in which correlation was very poor, resulted in substantial improvement in the remainder. It is suggested that in the central regions of large "doughnut" infarcts, reduced blood flow hinders the efflux of CKMB from the centre causing an underestimate of infarct size. Pyrophosphate scintigraphy appears to be more accurate than CKMB release in measuring the size of these large anterior infarcts.

Adult↗
PubMed1982

Pyrophosphate heart scan in patients with progressive systemic sclerosis.

Scintigraphic examination of the myocardium, using 99mTc-labelled pyrophosphate, was carried out in 17 patients suffering from systemic sclerosis. This connective tissue disorder very often affects the myocardium secondarily. The results of the cardiac scan were compared with the information obtained from the electrocardiogram of systolic time intervals. In addition, spirometry was undertaken to detect a potential relation between cardiac and pulmonary involvement. The scan was found to be positive in seven patients and electrocardiographic findings were pathological in five patients only. The systolic time intervals were abnormal in three patients only. A ventilation disturbance was recorded in 10 patients. No clear relation was found between the results of the individual examinations. It is concluded that pyrophosphate heart scintigraphy may detect myocardial impairment in some cases of systemic sclerosis before it is manifested by heart failure. Examination of systolic time intervals is of little importance.

Adult↗