Incorporation Of Pyrophosphate Research Articles

Synthesis, structure and magnetic properties of a binuclear Co(II)-pyrophosphate complex, [Co2(phenanthroline-dione)4(P2O7)

A series of Co(II)-1,10-phenanthroline-5,6-dione (phendione) complexes were prepared as scaffolds for the incorporation of pyrophosphate or methylenediphosphonate bridging ligands. Minor modifications in reaction conditions yielded the mononuclear complexes [Co(phendione)(H2O)4](SO4)·2H2O (1) and methylenediphosphonate complex [Co(phendione)(H2O3PCH2PO3H)(H2O)2]Cl·3H2O (2). A bridged binuclear complex, [Co2(phendione)4(P2O7)]·nH2O (3) was also prepared and structurally characterized. The magnetic properties of 3 were investigated.

Pyrophosphate and Methylenediphosphonate Incorporated Uranyl Peroxide Cage Clusters

Six uranyl peroxide cage clusters containing 18–54 uranyl ions, as well as 6–27 pyrophosphate or methylenediphosphonate ligands, have been synthesized and characterized. These uranyl peroxide clusters self-assemble and crystallize from aqueous solutions containing uranyl nitrate, hydrogen peroxide, and pyrophosphate or methylenediphosphonate over a pH range from 5.3 to 8.1. In their structures, uranyl hexagonal bipyramids are linked by bridging peroxide, hydroxide, and pyrophosphate or methylenedisphosphonate ligands. The incorporation of pyrophosphate or methylenedisphosphonate results in several interesting features in the coordination of uranyl ions and the topology of the uranyl peroxide clusters.

Massively parallel pyrosequencing in HIV research

1411–1415Keywords: DNA sequence, homogeneous PCR products, pyrosequencingThe new massively parallel sequencing methods are soastonishing that one wonders whether space aliens aresecretly behind them. One technician, running a singleinstrument,canobtainuptoapproximately1billionbasesof DNA sequence in a few days. Here we describe thenew sequencing methods, briefly present a few appli-cations in HIV research, and then speculate on futuredirections.

Spectroscopic Characterization of Pyrophosphate Incorporation during Extraction of Peat Humic Acids

AbstractExtraction procedures represent a crucial point in the study of the molecular structure of humic substances (HS). Although alkaline extractants such as NaOH plus Na4P2O7 are usually employed for the extraction of the HS from soil and peat, little information concerning the interaction between extractants and humic acids is available. In this study, the results on the molecular structure of humic acids (HA) extracted and separated from a Sphagnum Irish peat sample with an alkaline pyrophosphate solution (0.1 M NaOH + 0.1 M Na4P2O7) are reported. The HA were redissolved in 5 mM NaCl, fractionated in six cut‐off fractions (nominal molecular weight: HA >300, 100–300, 50–100, 20–50, 10–20, and 5–10 kDa) using a tangential ultrafiltration system. All samples were dialyzed against distilled water and then freeze‐dried. Fourier‐transform infrared (FT‐IR) and 31P nuclear magnetic resonance (NMR) spectroscopies were applied to characterize the incorporation of pyrophosphate (PP) in both the unfractionated HA and in the six HA fractions. Spectroscopic analyses showed that it was possible to assign a given pattern of bands to define the structure of phosphate: PP was present only in the unfractionated HA, whereas orthophosphate was present in both the high molecular weight and lower molecular weight fractions. The high concentration of phosphate found in the lower molecular weight HA fractions has been attributed to the presence of metals and the oxygenated groups of HA. The changes in the relative intensity of the bands attributed to oxygenated functional groups observed in the FT‐IR spectra could be assigned to the interaction of phosphate groups with metal cations, which are involved in a coordination complex with the oxygenated groups.

Effects of reperfusion intervals on skeletal muscle injury beneath and distal to a pneumatic tourniquet

To date there have been no experimental studies specifically directed at effects of reperfusion intervals on skeletal muscle injury beneath the tourniquet. 99mTechnetium pyrophosphate (Tc 99) incorporation and correlative histology were used to assess injury 2 days after tourniquet application in muscles beneath (thigh) and distal (leg) to the cuff. Tourniquets were applied to rabbit hindlimbs for a total of either 2 or 4 hours. In the 4-hour series, tourniquet compression (either 125 mm Hg or 350 mm Hg cuff inflation pressure) was either continuous or interrupted by 10-minute reperfusion intervals after 2 hours or after every hour of cuff inflation. In the 2-hour series, tourniquet compression (350 mm Hg) was either continuous or interrupted by a 10-minute reperfusion interval after 1 hour. Pyrophosphate incorporation (Tc 99 uptake) was significantly greater in the thigh region than in the leg region in all of the 4-hour tourniquet groups. Tc 99 uptake was significantly reduced by reperfusion after each hour of cuff inflation. With 350 mm Hg tourniquet pressure, a reperfusion interval after 2 hours of cuff inflation tended to exacerbate tourniquet compression injury. Reperfusion intervals did not significantly affect Tc 99 uptake in the leg region of these groups. With a 2-hour tourniquet time, Tc 99 uptake in the thigh was significantly decreased by reperfusion after 1 hour of cuff inflation. Previous clinical recommendations, based on serum creatine phosphokinase abnormalities after experimental tourniquet ischemia, probably reflected tourniquet compression injury. Hourly reperfusion limits skeletal muscle injury during extended periods of tourniquet use.

Muscle injury induced beneath and distal to a pneumatic tourniquet: A quantitative animal study of effects of tourniquet pressure and duration

Previous recommendations regarding the “safe” period of tourniquet hemostasis were based largely on studies of ischemia distal to the tourniquet. This study quantitatively analyzed skeletal muscle injury induced beneath and distal to a pneumatic tourniquet applied to the hindlimbs of rabbits for 1, 2, or 4 hours with a cuff inflation pressure of 125, 200, or 350 mm Hg. Technetium Tc 99m pyrophosphate incorporation after systemic injection (Tc 99 uptake) and correlative histology were used to evaluate tissue damage 2 days after tourniquet application. Compared with the contralateral control limbs, compression and ischemia induced statistically significant increases in Tc 99 uptake in the thigh and leg regions of all groups. Pyrophosphate incorporation was significantly greater in the thigh region than in the leg region after 2 hours of compression in the 200 and 350 mm Hg pressure groups and following 4 hours of compression in all pressure groups. Focal and regional fiber necrosis and degeneration were observed in thigh muscles after 2 hours of tourniquet compression. Two hours of continuous tourniquet application at clinically relevant cuff inflation pressures induced significant skeletal muscle necrosis beneath the tourniquet. Use of the lowest possible inflation pressure for a limited duration should minimize the degree of tissue injury caused by tourniquet application.

Purification of a Prenyltransferase That Elongates cis-Polyisoprene Rubber from the Latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.

Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium argentatum Gray) by Low Temperature and 2-(3,4-Dichlorophenoxy) Triethylamine

Rubber particles isolated from guayule (Parthenium argentatum Gray) stem homogenates contain a polyprenyl transferase which catalyzes the polymerization of isopentenyl pyrophosphate into polyisoprene. The polymerization reaction is stimulated with the addition of an allylic pyrophosphate initiator and forms a polymer of polyisoprene with a molecular weight distribution from 10(3) to 10(7). The polymerization reaction in crude stem homogenates is not affected by the addition of an initiator probably due to the high activity of isopentenyl pyrophosphate isomerase furnishing saturating levels of dimethylallyl pyrophosphate. Polyisoprene formation in stems of guayule plants exposed to cold winter temperatures increased from 15.4 milligrams per gram dry weight in October to 24.5 milligrams per gram dry weight in January and increased from 16.2 to 38.1 milligrams per gram dry weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4-dichlorophenoxy)triethylamine. The rate of polymerization of isopentenyl pyrophosphate into polyisoprene in stem homogenates of the cold treated plants increased from 12.1 nanomoles per hour per gram fresh weight in October to 144.3 nanomoles per hour per gram fresh weight in January and increased from 17.7 to 446.8 nanomoles per hour per gram fresh weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4-dichlorophenoxy)triethylamine. These results show that the increase in polyprenyl transferase activity partially accounts for the increase in polyisoprene synthesis in guayule plants exposed to low temperature and treated with 2-(3,4-dichlorophenoxy)triethylamine.

A new method for radionuclide imaging of acute myocardial infarction in humans.

A new method of visualizing acute myocardial infarction in humans following intravenous injection of 15 mCi-5 mg of 99m Tc stannous pyrophosphate in 23 patients is reported. Fifteen patients had histories suggestive of acute myocardial infarction and subsequently developed electrocardiogram and enzyme changes that confirmed the clinical diagnosis. Eleven of the 15 patients were scanned 3-5 days postinfarction, and all had positive scintigrams. The four remaining patients were scanned 7-10 days after their myocardial infarction; two had positive scintigrams. In those 8 patients with chest pain but without ECG and enzyme changes suggestive of myocardial infarction, scintigrams were negative. Positive scintigrams in the patients with myocardial infarction are thought to be due to incorporation of pyrophosphate into the crystalline structure of the hydroxyapatite found within the mitochondria of irreversibly damaged myocardial cells. The location of the acute myocardial infarction by scintigram correlated well with ECG localization in the 13 patients with positive scintigrams. This imaging method shows promise in 1) identifying the presence of acute myocardial infarction in patients with chest pain, 2) determining the location of acute myocardial infarction with a high degree of accuracy, 3) detecting the extension of the infarction, and 4) the possibility of determining the size of acute myocardial infarctions.

The demonstration of cinnamyl-CoA synthetase activity in leaf extracts

Abstract Cinnamyl-CoA synthetase activity has been demonstrated in extracts of leaves of spinach beet ( Beta vulgaris L. ssp. vulgaris ) by direct spectrophotometric assay and by CoA-dependent formation of cinnamyl hydroxamate. These extracts also promoted a cinnamate-dependent incorporation of pyrophosphate into nucleotide phosphate, which was inhibited by CoA. The activity of extracts was affected by the age of the plants and leaves and by their growth conditions; it showed an inverse correlation with the accumulation of red pigment in the leaves. Measurable activity was also found in leaves from young plants of pea, runner bean and spinach.

A radioactive hydroxamate method for determining rates of amino acid activation

1. 1. Amino acid-activating enzymes which catalyze the reaction between α-amino acids and ATP to form aminoacyl adenylates can be assayed by colorimetric measurement of the rate of formation of the corresponding hydroxamates in the presence of hydroxylamine or by measurement of the incorporation of radioactive pyrophosphate into ATP. The first of these techniques is quite insensitive and rarely used for quantitative work. The second technique is reliable and sensitive but cannot be used if more than one substrate is being activated or if there are other reactions occurring which lead to incorporation of pyrophosphate into ATP. 2. 2. By “activating” [ 14 C]amino acids in the presence of hydroxylamine it is possible to measure the formation of [ 14 C]amino acid hydroxamate. By assaying for the amount of [ 14 C]hydroxamate formed one is able to measure the extent of activation of one particular substrate, the 14 C-labeled substrate, regardless of whether other substrates are being activated or whether there are other pyrophosphate-incorporating reactions. 3. 3. Experimental details are provided which permit rapid and simple separation of [ 14 C]amino acid from [ 14 C]hydroxamate on ion-exchange paper, accurate assay of relative radioactivities and accurate estimation of reaction rates. The techniques are shown to be useful both with purified enzymes and crude tissue extracts.

A radioactive hydroxamate method for determining rates of amino acid activation

Abstract 1. 1. Amino acid-activating enzymes which catalyze the reaction between α-amino acids and ATP to form aminoacyl adenylates can be assayed by colorimetric measurement of the rate of formation of the corresponding hydroxamates in the presence of hydroxylamine or by measurement of the incorporation of radioactive pyrophosphate into ATP. The first of these techniques is quite insensitive and rarely used for quantitative work. The second technique is reliable and sensitive but cannot be used if more than one substrate is being activated or if there are other reactions occurring which lead to incorporation of pyrophosphate into ATP. 2. 2. By “activating” [14C]amino acids in the presence of hydroxylamine it is possible to measure the formation of [14C]amino acid hydroxamate. By assaying for the amount of [14C]hydroxamate formed one is able to measure the extent of activation of one particular substrate, the 14C-labeled substrate, regardless of whether other substrates are being activated or whether there are other pyrophosphate-incorporating reactions. 3. 3. Experimental details are provided which permit rapid and simple separation of [14C]amino acid from [14C]hydroxamate on ion-exchange paper, accurate assay of relative radioactivities and accurate estimation of reaction rates. The techniques are shown to be useful both with purified enzymes and crude tissue extracts.