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At least 721 records · Page 40Linked to original sources
PubMed2002

Inactivation of the human brain muscarinic acetylcholine receptor by oxidative damage catalyzed by a low molecular weight endogenous inhibitor from Alzheimer's brain is prevented by pyrophosphate analogs, bioflavonoids and other antioxidants.

Oxidative stress has been implicated as a contributing factor to neurodegeneration in Alzheimer's disease. An endogenous, low molecular weight (LMW) inhibitor from Alzheimer's brain inactivates the human brain muscarinic acetylcholine receptor (mAChR). The inhibitor prevents agonist and antagonist binding to the mAChR as assessed by radioligand binding studies. The LMW endogenous inhibitor, which has components with molecular weights between 100 and 1000 Da, requires dissolved oxygen and glutathione. Prevention of inactivation of the mAChR with peroxidase suggests that the LMW endogenous inhibitor generates peroxide. Heme, previously shown to be present in the LMW endogenous inhibitor, also inactivates the mAChR in the presence of peroxide. Free radical damage to the muscarinic receptor by the endogenous inhibitor can be prevented through the use of naturally occurring antioxidants including bilirubin, biliverdin, carnosol, myricetin and quericetin. In addition, pyrophosphate, imidodiphosphate, bisphosphonates and related compounds also protect the muscarinic receptor from free radical damage. Inactivation of the mAChR by the LMW endogenous inhibitor is likely to be a factor in the continual decline of Alzheimer's patients, even those taking acetylcholinesterase inhibitors. Natural antioxidants and pyrophosphate analogs may improve the effectiveness of acetylcholinesterase inhibitors and prove useful in the treatment and prevention of Alzheimer's disease since the muscarinic acetylcholine receptor is required for memory, and decreased cholinergic function is a critical deficit in Alzheimer's disease.

Alzheimer Disease↗
PubMed1977

99Tcm-Sn pyrophosphate scanning in suspected acute myocardial infarction using a mobile rectilinear scanner.

99Tcm stannous pyrophosphate injected intravenously gives a positive image of freshly damaged myocardium but not of normal or scarred myocardium. It is safe, cheap and generally available. One hundred and forty patients admitted to hospital with possible myocardial infarction were scanned with a mobile rectilinear scanner of the type available in most district general hospitals. When the diagnosis of infarction was definite on clinical, electrocardiographic and enzyme criteria the anterior scan was positive in 31 out of 36 patients (86%); and when it was probable, the scan was positive in 28 out of 41 (68%) and when it was doubtful the scan was positive in 23 out of 63 (37%). The optimum time for scanning was between the second and seventh days. Pyrophosphate scanning is a very valuable investigation even though it is not an absolute discriminator of myocardial infarction. It is useful in assessing patients with atypical or doubtful symptoms of infarction where the ECG is already abnormal or where there are other causes of raised enzyme concentrations. False negative scans are not common, but a negative scan does not outweigh strong alternative evidence of infarction. Positive scans occur in some patients with unstable angina without confirmatory evidence of infarction. Positive scans due to extracardiac lesions are distinguished readily.

Humans↗
PubMed2001

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

Bacterial Proteins↗
PubMed1999

Evidence that fructose 1,6-bisphosphate specifically protects the alpha-subunit of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase against proteolytic degradation.

Pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) consists of alpha (regulatory) and beta (catalytic) subunits. The alpha-subunit was previously reported to be much more susceptible to tryptic digestion than the beta-subunit. In this study, ligand-induced protection of PFP subunits against proteolysis by subtilisin was investigated in vitro and the data obtained demonstrated that fructose 1,6-bisphosphate (Fru-1,6-P(2)), while exerting negligible effect on the beta-subunit, remarkably protected the alpha-subunit against proteolytic degradation. Western blot analysis revealed a good correlation between the Fru-1,6-P(2) concentration and the degree of corresponding protection on the alpha-subunit against proteolysis. In contrast, none of other examined ligands including fructose 2,6-bisphosphate, fructose 6-phosphate and pyrophosphate had such protection on the alpha-subunit. This finding (1) indicates that the stability of the alpha-subunit can be selectively increased by Fru-1,6-P(2), and (2) suggests that Fru-1,6-P(2) is likely a special effector of the alpha-subunit.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Transcriptional regulation of farnesyl pyrophosphate synthase by liver X receptors.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that are involved in cholesterol and lipid metabolism. In addition to liver, the brain is another site where LXRs may control cholesterol homeostasis. In the brain, the regulation of cholesterol homeostasis is independent from other parts of the body, and its disturbance is associated with neurodegenerative disorders, such as Alzheimer's disease. We have used PCR-based suppressive subtractive cloning to identify new LXR target genes in brain cells. In this report, we show that farnesyl pyrophosphate synthase (FPPS) is a new target gene for LXR in astrocytes and neurons. Farnesyl pyrophosphate is an obligate intermediate for de novo cholesterol synthesis and a substrate for protein farnesylation. Stimulation of FPPS mRNA synthesis by an LXR agonist, Hypocholamide, was observed in several cell lines from the central nervous system. We identified a single putative direct repeat 4 (DR4) LXR response element in the FPPS promoter. In a reporter gene assay, LXR transactivated a reporter gene bearing a truncated FPPS promoter containing this DR4 cis-element but not if the DR4 element was mutated. Using gel-mobility shift assay, we further demonstrated the direct interaction between the LXR/retinoid X receptor (RXR) heterodimer and the response element. Taken together, our results indicate that LXRs directly regulate FPPS gene expression, and thus may play a role in modulating cholesterol synthesis in the brain.

Alkyl and Aryl Transferases↗
PubMed1981

Effect of intra-articular yttrium-90 on chronic pyrophosphate arthropathy of the knee.

Fifteen patients with bilateral, symmetrical, chronic pyrophosphate arthropathy of the knee were given intra-articular injections of yttrium-90 (5 mCi) plus steroid (triamcinolone hexacetonide, 20 mg) into one knee, and saline plus steroid into the other (control) knee. Allocation of the 90Y injection was random and double blind. After 6 months there was significantly less pain, inactivity stiffness, joint-line tenderness, and effusion in the 90Y-injected knees than in the controls (p less than 0.01). There were also significant differences between 90Y-injected and control knees in the changes in range of movement (p less than 0.01) and joint circumference (p less than 0.05) caused partly by progression of disease in the control knees. No significant differences in joint deformity, instability, X-ray appearance, or synovial-fluid analysis were seen. In all cases patient and observer assessment favoured the treated side (p less than 0.01). These findings indicate that intra-articular 90Y may be of benefit in chronic pyrophosphate arthropathy, a disease for which there is no treatment. The predilection of this condition to affect the knees of the elderly makes such treatment highly suitable because the joint lends itself readily to injection and the procedure carries very few actual or potential risks in this age group.

Aged↗
PubMed2002

Vgamma9Vdelta2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens.

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of gammadelta T cells, predominantly Vgamma9Vdelta2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the gammadelta T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-gamma but no IL-4. By single-cell analysis of intracellular IFN-gamma production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4(-)CD8(-) gammadelta T cells. The data presented suggest the infiltration of PPP-specific Vgamma9Vdelta2 Th1-like cells into the eye of BD patients with uveitis.

Adult↗
PubMed1997

X-ray characteristics of wrists in calcium pyrophosphate crystal deposition disease. Is pseudogout a major cause of scapholunate advanced collapse?

Deposition of calcium pyrophosphate dihydrate (CPPD) crystals has been considered to be a cause of scapholunate advanced collapse (SLAC) wrist. The aim of this study was to look at X-ray changes in wrist joints affected by CPPD crystal deposition disease and to determine whether crystal deposition is a cause of SLAC wrist. A total of 150 wrists of 78 patients with CPPD crystal deposition disease were examined. In our population of Japanese patients with CPPD crystal deposition disease, the incidence of SLAC wrist was very low, and no case of Stage III SLAC wrist was found. We therefore conclude that SLAC wrist is not a radiographic characteristic of CPPD crystal deposition disease and that pyrophosphate crystal deposition cannot be a major cause of SLAC wrist.

Aged↗
PubMed2003

Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates Its potential to activate human gammadelta T cells but does not lead to competitive antagonism.

The immunological characterization of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), and its methylenediphosphonate analogue, HMB-PCP, is described. With an EC(50) of 0.1-0.2 nM, HMB-PP is significantly more potent in stimulating human Vgamma9/Vdelta2 T cells than any other compound described so far. However, replacing the pyrophosphate by a P-CH(2)-P function abrogates the bioactivity drastically, with HMB-PCP having a EC(50) of only 5.3 microM.

Binding, Competitive↗
PubMed1998

Phosphonate and bisphosphonate analogues of farnesyl pyrophosphate as potential inhibitors of farnesyl protein transferase.

Several phosphonate and bisphosphonate analogues of farnesyl pyrophosphate have been prepared for an examination of their ability to inhibit farnesyl protein transferase (FPTase). A Horner-Wadsworth-Emmons condensation of farnesal or geranial with tetraethyl methylenediphosphonate gave the desired vinyl phosphonates, while alkylation of the dimethyl methylphosphonate anion with a terpenoid bromide gave the corresponding saturated phosphonates. Alkylation of tetraethyl methylenediphosphonate with farnesyl bromide gave the expected alkyl bisphosphonate, which was converted to its alpha, beta-unsaturated derivative by preparation of the phenyl selenide, oxidation to the selenoxide, and elimination. In a similar fashion, triethyl phosphonoacetate was converted to a farnesyl pyrophosphate analogue by reaction with farnesyl bromide. After preparation of the respective acids, each compound was tested for inhibition of FPTase at concentrations ranging up to 10 microM. The effect of these compounds on FPTase activity varied substantially, ranging from depressed to surprisingly enhanced enzymatic activity.

Alkyl and Aryl Transferases↗
PubMed1995

Calcium pyrophosphate dihydrate crystal deposition disease: a review of the literature and a light and electron microscopic study of a case of the temporomandibular joint with numerous intracellular crystals in the chondrocytes.

The pathogenesis of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease of synovial joints is still unclear, although overproduction of extracellular pyrophosphate (PPi) is thought to play a key role. We studied the light and electron microscopic appearances of a case of CPPD crystal deposition disease of the temporomandibular joint (TMJ) in search of new clues for its pathogenesis. Light microscopic examination of CPPD-containing material from the joint space revealed cartilaginous nodules with various degrees of crystallization. Transmission electron microscopic examination revealed numerous extra- as well as intracellular crystals and crystal shaped spaces in the chondrocytes. Other striking ultrastructural features of the chondrocytes included the presence of many mitochondria, frequently containing crystalline material, and the presence of highly dilated rough endoplasmic reticulum and large glycogen islands. The presence of intramitochondrial crystals may hypothetically imply a derangement in mitochondrial adenosine triphosphate or PPi metabolism. The finding of intracellular CPPD crystals in chondrocytes points to the existence of an intracellular pathway of CPPD crystal formation in CPPD crystal deposition disease of the TMJ and possibly in CPPD crystal deposition disease in general.

Cartilage, Articular↗
PubMed1994

Insulin-like growth factor-1 suppresses pyrophosphate elaboration by transforming growth factor beta1-stimulated chondrocytes and cartilage.

Our objective was to examine the effect of insulin-like growth factor-1 (IGF-1) on extracellular pyrophosphate (ePPi) elaboration by porcine cartilage. These studies further define the factors influencing ePPi accrual, a key step in calcium pyrophosphate dihydrate (CPPD) crystal formation. ePPi was measured in adult porcine organ and monolayer culture media in the presence of IGF-1, transforming growth factor beta-1 (TGFbeta-1), IGF-1 antibody and synovial fluid (SF). As previously shown, TGFbeta-1 stimulated ePPi elaboration by cartilage and chondrocytes. IGF-1 significantly inhibited the stimulatory effect of TGFbeta-1 on ePPi elaboration by both cartilage explants and chondrocytes. Anti-IGF-1 antibody blocked this inhibition. Anti-IGF-1 antibody also decreased the inhibitory effect of SF on ePPi elaboration, suggesting the presence of active IGF-1. These results support an important regulatory role for IGF-1 in cartilage ePPi elaboration. IGF-1 inhibited the effects of the ePPi-stimulatory factor TGFbeta-1 and thus may protect normal joints from excess accumulation of ePPi and subsequent CPPD crystal formation.

Animals↗
PubMed1991

The substrate reactivity of mu-monothiopyrophosphate with pyrophosphate-dependent phosphofructokinase: evidence for a dissociative transition state in enzymatic phosphoryl group transfer.

mu-Monothiopyrophosphate (MTP), an analogue of pyrophosphate (PPi) with sulfur in place of oxygen in the bridge position, is a substrate for the enzyme pyrophosphate-dependent phosphofructokinase. At pH 9.4 and 6 degrees C, the maximal velocity for the phosphorylation of fructose 6-phosphate (F6P) by MgMTP is about 2.8% of that with MgPPi as the phosphoryl donor. The kinetic mechanism is equilibrium random with rate-limiting transformation of the substrate ternary complex to the product when either MgMTP or MgPPi is the phosphoryl donor. This is known from independent studies to be kinetic mechanism at pH 8.0 and 25 degrees C [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101-4108]. The dissociation constant of MgPPi is 14 microM, that of MgMTP is 64 microM, and that of F6P from the enzyme is about 5 mM. The Km values for MgPPi and MgMTP are 14.5 and 173 microM, respectively. MgMTP competes with MgPPi for binding to the enzyme. The values of kcat are 3.4 s-1 and 140 s-1 for MgMTP and MgPPi, respectively, at pH 9.4 and 6 degrees C. The estimated rate enhancement factors are 3.6 x 10(5) and 1.4 x 10(14) for the reactions of MgMTP and MgPPi, respectively. Therefore, MgMTP is a reasonably good substrate for PPi-dependent PKF, on the basis of comparisons of kcat. However, the rate enhancement factors show that the enzyme is a poor catalyst for the reaction of MgMTP. Lesser enzymatic catalysis in the reaction of MgMTP compared with MgPPi is largely compensated for by the greater intrinsic reactivity of MgMTP. Thus, the larger substrate MgMTP is well accommodated in the active site, and the dissociative reaction of MgMTP is well accommodated in the transition state. The results are interpreted to indicate a dissociative transition state for phosphoryl group transfer by PPi-dependent PFK. A modified synthesis and purification of MTP are described, in which (trimethylsilyl)trifluoromethanesulfonate and tetra-N-butylammonium iodide are used in place of iodotrimethylsilane to dealkylate tetramethyl-MTP.

Animals↗
PubMed1991

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.

Adenosine Triphosphate↗
PubMed1982

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase.

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.

Animals↗
PubMed1986

Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases.

Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrates↗
PubMed1981

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid.

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.

Adenosine Diphosphate↗
PubMed1981

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration.

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.

Chromatography, Thin Layer↗