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PubMed1967

The purification of 3,3-dimethylallyl- and geranyl-transferase and of isopentenyl pyrophosphate isomerase from pig liver.

The enzyme catalysing the synthesis of farnesyl pyrophosphate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate, or from geranyl pyrophosphate and isopentenyl pyrophosphate, has been purified 100-fold from homogenates of pig liver. The enzyme has optimum pH 7.9 and requires Mg(2+) as activator in preference to Mn(2+); it is inhibited by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate and phosphate ions in addition to the products of the reaction, inorganic pyrophosphate and farnesyl pyrophosphate. From product-inhibition studies of the geranyltransferase reaction, the order of addition of substrates to and release of products from the enzyme has been deduced: geranyl pyrophosphate combines with the enzyme first, followed by isopentenyl pyrophosphate. Farnesyl pyrophosphate dissociates from the enzyme before inorganic pyrophosphate. The existence of isopentenyl pyrophosphate isomerase in liver is confirmed. Methods for the preparation of the pyrophosphate esters of isopentenol, 3,3-dimethylallyl alcohol, geraniol and farnesol are also described.

Animals↗
PubMed1976

Bone pyrophosphate in uremia and its association with extraosseous calcification.

The mean bone pyrophosphate was 0.360 +/- 0.15 mg/g in 8 controls and 1.22 +/- 1.39 mg/g bone in 27 uremic patients (P less than 0.0025). 13 of the 27 uremic patients had bone pyrophosphate levels greater than 2 SD above control values. The ash content of uremic bones with increased pyrophosphate levels (group II) was 56 +/- 9% as compared to 64 +/- 2% in control bones (P less than 0.01) and 60 +/- 7% in uremic bones having normal pyrophosphate levels (P less than 0.1) (group I). The magnesium content of bones in group II was 338 +/- 47 as compared to 211 +/- 13 (P less than 0.0005) in the controls and 294 +/- 73 mmol/kg ash (P less than 0.05) in group I. In group II, but not group I, there was a significant inverse correlation between duration of dialysis and percent bone ash (r = -0.59) (P less than 0.05). A definite relationship existed between elevated bone pyrophosphate levels and soft tissue calcification. In group II the mean pulmonary calcium content was 530 +/- 459 as compared to 32 +/- 26 mmol/kg/ash in group I (P less than 0.0025). All patients with a bone pyrophosphate level greater than 1.4 mg/g bone had extensive pulmonary calcification. It is concluded that the excess bone pyrophosphate present in some uremic patients is either deposited in the apatite crystal in the transphosphorylated form or else as the magnesium salt since the pyrophosphate is resistant to pyrophosphatase and surface adsorption of pyrophosphate is not altered by the increased bone pyrophosphate levels. The excess bone pyrophosphate could disturb bone calcification mechanisms in uremic patients. The association between increased bone pyrophosphate and soft tissue calcification suggests that the disordered pyrophosphate metabolism may be important in the pathogenesis of extraosseous calcification.

Binding Sites↗
PubMed1969

Mechanism of squalene biosynthesis: evidence against the involvement of free nerolidyl pyrophosphate.

Several mechanisms that utilize farnesyl pyrophosphate and nerolidyl pyrophosphate as condensing substrates have been postulated for the asymmetric condensation reaction in squalene biosynthesis. Although there is ample evidence that farnesyl pyrophosphate is a substrate for this reaction, there has been no information concerning the role of nerolidyl pyrophosphate. We have made the following observations that demonstrate that nerolidyl pyrophosphate cannot be a free intermediate in squalene biosynthesis. (a) There is no significant interconversion of farnesyl pyrophosphate and nerolidyl pyrophosphate in a squalene-synthesizing system from yeast. (b) Nerolidyl-1-(3)H(2) pyrophosphate is not converted to squalene in the presence or absence of farnesyl pyrophosphate. (c) The addition of unlabeled nerolidyl pyrophosphate to incubation mixtures does not alter the relative loss of alpha-hydrogens from farnesyl pyrophosphate during its conversion to squalene. The synthesis of nerolidyl-1-(3)H(2) pyrophosphate is described. Chromatographic methods for the separation of pyrophosphate esters of triprenols and terpenols are included.

Carbon Isotopes↗
PubMed1995

Synthesis of medium pyrophosphate by soluble mitochondrial F1 through dimethyl sulfoxide-water transitions.

Soluble F1 from heart mitochondria incubated in mixtures that have Mg2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., García, J. J., and Gómez-Puyou, A. (1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration experiments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PPi in F1 showed that hydrolysis of synthesized PPi at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PPi but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F1 that had been depleted of adenine nucleotides. Hence, dilution-induced release of PPi was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium Pi and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F1.

Adenosine Triphosphate↗
PubMed1987

Is pyrophosphate an analog of adenosine diphosphate for beef heart mitochondrial F1-ATPase.

Beef heart mitochondrial F1 possesses three pyrophosphate-binding sites, which comprises one high affinity binding site (Kd approximately equal to 1 microM) and two lower affinity sites (Kd approximately equal to 20 microM). High affinity pyrophosphate binding required the presence of Mg2+ in the incubation medium. Pyrophosphate competed with ADP, but not with Pi for binding to mitochondrial F1. Upon binding of 3 mol of pyrophosphate/mol of F1, one of the three tightly bound nucleotides present in native F1 was released. Like ADP and in contrast to Pi, pyrophosphate enhanced the fluorescence intensity of F1-bound aurovertin, and it prevented the photolabeling of F1 by 2-azido-ADP. As aurovertin and 2-azido-ADP are ligands of the beta subunit of F1, it is likely that pyrophosphate binds preferentially to the beta subunit. Whereas the binding affinity of F1 for Pi was increased by concentrations of pyrophosphate lower than 100 microM, it was decreased by a higher concentration of pyrophosphate. This biphasic effect of pyrophosphate on Pi binding was not observed with ADP, which, at all concentrations tested, inhibited Pi binding. Except for the effect of pyrophosphate on Pi binding to F1, for all the other effects, pyrophosphate mimicked ADP. It is suggested that pyrophosphate and ADP share the same binding site on F1 and that pyrophosphate interacts with the same amino acid residues as those interacting with the alpha and beta phosphate groups of ADP.

Adenosine Diphosphate↗
PubMed1972

Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores.

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.

Adenosine Diphosphate↗
PubMed1990

The relationship between the thiamin pyrophosphate effect and the saturation status of the transketolase with its coenzyme in human erythrocytes.

The thiamin pyrophosphate effect has been used as a reliable index to evaluate the nutritional status of thiamin. But there has not been any report concerning whether or not the thiamin pyrophosphate effect really reflects the saturation status of transketolase with thiamin pyrophosphate. In this report we studied the relationship between the thiamin pyrophosphate effect and the saturation status of transketolase. First, we determined the thiamin pyrophosphate concentrations, transketolase activities, thiamin pyrophosphate effects, and transketolase concentrations in human hemolysates from 16 apparently healthy subjects. The molar ratio of thiamin pyrophosphate to transketolase was in inverse proportion to the thiamin pyrophosphate effect. Second, we prepared apotransketolase preparations and reconstituted it with various concentrations of thiamin pyrophosphate. The thiamin pyrophosphate effects in these preparations were in good correspondence with the ratios of apotransketolase. These results indicate that the thiamin pyrophosphate effect really reflects the saturation status of transketolase with coenzyme.

Adult↗
PubMed1989

Monoterpene biosynthesis: mechanistic evaluation of the geranyl pyrophosphate:(-)-endo-fenchol cyclase from fennel (Foeniculum vulgare).

Geranyl pyrophosphate:(-)-endo-fenchol cyclase catalyzes the conversion of geranyl pyrophosphate to (-)-endo-fenchol by a process thought to involve the initial isomerization of the substrate to the tertiary allylic isomer, linalyl pyrophosphate, and the subsequent cyclization of this bound intermediate. Studies with 18O-labeled acyclic precursors and H2(18)O, followed by mass spectrometric analysis of the cyclic product, confirmed that water was the sole source of the carbinol oxygen atom of endo-fenchol, thus indicating the participation of the solvent in terminating this presumptive carbocationic reaction. The isomerization component of the normally coupled reaction sequence was demonstrated directly using the substrate analog 2,3-cyclopropylgeranyl pyrosphosphate and by isolating the corresponding homoallylic analog of linalyl pyrophosphate as a major reaction product. The cyclization component of the reaction sequence was effectively dissected using linalyl pyrophosphate as substrate, and both isomerization and cyclization steps were shown to take place at the same active site of the cyclase, an observation consistent with the efficient coupling of these processes. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate were shown to be effective inhibitors of the cyclase, and the electron-withdrawing substituent was shown to greatly suppress the rate of cyclization of these labeled analogs, indicating that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate. Additional evidence for the electrophilic nature of the reaction was obtained by demonstrating the ability of the cyclase to solvolyze other substrate analogs which bear an allylic pyrophosphate, and by showing that cyclization was strongly inhibited by sulfonium analogs of presumptive carbocationic intermediates of the reaction sequence, especially in the presence of inorganic pyrophosphate as counterion. In spite of the fact that the fenchol cyclase terminates the cyclization with an external nucleophile (H2O), the primary mechanistic features of this isomerization-cyclization reaction are similar to those catalyzed by other cyclases that terminate the reaction by deprotonation or cation capture by the pyrophosphate moiety of the substrate.

Intramolecular Lyases↗
PubMed1998

Crucial role of TCR gamma chain junctional region in prenyl pyrophosphate antigen recognition by gamma delta T cells.

Human gamma delta T cells recognize prenyl pyrophosphate Ags and their analogues in a V gamma 2V delta 2 TCR-dependent manner. Few data are available regarding the TCR structural requirements for recognition of such prenyl pyrophosphate Ags by gamma delta T cells. Presently, we made chain pair switch, chimeric, and site mutant gamma delta TCRs and transfected them into TCR- mutant Jurkat T cells to examine the effects of changing the TCR gamma junctional region sequences on reactivity to prenyl pyrophosphate Ags. Substitution of the TCR gamma junctional region (N and J) sequences from an Ag-reactive TCR with TCR gamma junctional region sequences from an Ag-nonreactive TCR abrogated reactivity to the prenyl pyrophosphate Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate but not to a mycobacterial supernatant containing a mixture of prenyl pyrophosphate Ags. Substitution of only the TCR gamma N nucleotide region with that from this Ag-nonreactive TCR destroyed reactivity to isopentenyl pyrophosphate and to the mycobacterial supernatant. Substitution of the entire V delta 2 chain from the Ag-reactive TCR with a V delta 1 chain from an Ag-nonreactive TCR yielded a prenyl pyrophosphate Ag-nonreactive TCR. Thus, using TCR mutagenesis and TCR transfectants, we show that gamma delta TCR reactivity to prenyl pyrophosphate Ags is dependent upon the junctional region of the TCR gamma chain and upon pairing of V gamma 2 and V delta 2 TCR chains. These structural requirements of TCR gamma delta recognition of prenyl pyrophosphates distinguish this reactivity from that of protein superantigens and emphasize the importance of the TCR gamma CDR3 loop and adjacent residues.

Amino Acid Sequence↗
PubMed1986

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction.

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.

Catalysis↗
PubMed1988

Urinary pyrophosphate in patients with recurrent calcium urolithiasis and in healthy controls: a re-evaluation.

The excretion of inorganic pyrophosphate was studied in daily, fasting and postprandial urine specimens of normocalciuric and hypercalciuric patients with recurrent renal calcium stone disease (40 men and 40 women), and healthy controls (20 men and 20 women). Both populations were subdivided into younger (20 to 40 years old) and older (more than 40 years old) individuals. In general, there was a tendency towards higher urinary pyrophosphate excretion with increasing age (both sexes and all groups studied), and lower excretion in women than in men. The urinary pyrophosphate excretion rate was unchanged in daily and fasting urine specimens of the younger male normocalciuric and idiopathic hypercalciuric stone patients, whereas in the daily and postprandial urine of younger women the median excretion rate was reduced (controls versus normocalciuric plus idiopathic hypercalciuric subjects, 3 versus 1 mumol., p less than 0.05). In contrast, in older men urinary pyrophosphate was reduced in daily specimens (controls versus normocalciuric plus idiopathic hypercalciuric subjects, 55 versus 33 mumol., p less than 0.05) but it was unchanged in fasting urine specimens. In older women no change was detectable in any of the 3 urine portions. Factorization of urinary pyrophosphate for the associated urinary creatinine did not alter these results substantially, and the presence of renal stones did not modify pyrophosphate excretion significantly. Urinary pyrophosphate was correlated significantly with urinary volume, citrate and phosphorus. We conclude that only subclassification of stone patients with respect to sex, age and type of calciuria, and consideration of additional urine portions besides the daily urine may help to uncover states of urinary pyrophosphate deficit. On the basis of the data, we recommend that clinically relevant studies on inhibitory effects of urinary pyrophosphate on the nucleation and growth of crystals and stones should be done preferentially in urine portions with a proved pyrophosphate deficit.

Adult↗
PubMed1999

Pyrophosphate increases the efficiency of enterobactin-dependent iron uptake in Escherichia coli.

Exogenous inorganic pyrophosphate increases the biomass yield of Escherichia coli. In this report, we show that the effect of pyrophosphate is related to iron uptake. We have found that addition of pyrophosphate, ammonium iron (III) citrate or iron (III) chloride, in M63 minimal medium containing 1.7 microM of iron, causes an increase in growth yield. In contrast to iron chloride or ammonium iron (III) citrate, exogenous pyrophosphate is deleterious to strains unable to synthesize enterobactin. Thus the positive effect of pyrophosphate is related to the enterobactin uptake system expressed in a low iron content medium. Pyrophosphate in minimal medium has a repressing effect on the expression of Fur-regulated genes. In iron rich medium where enterobactin synthesis is strongly decreased, addition of pyrophosphate increases expression of Fur-regulated genes. Furthermore, this latter regulatory effect of pyrophosphate in iron-rich medium is enhanced in the absence of enterobactin synthesis. It has also been shown that addition of pyrophosphate protects the cell against the oxidative stress caused by the presence of hydrogen peroxide in an iron-rich containing medium. These results indicate that pyrophosphate acts as an iron-chelating agent, could trigger the enterobactin-dependent iron uptake system and could promote an increased binding of iron to enterobactin.

Base Sequence↗
PubMed2003

Vacuole membrane topography of the DPP1-encoded diacylglycerol pyrophosphate phosphatase catalytic site from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the beta-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane.

Amino Acid Motifs↗
PubMed1985

Isoprenoid enzyme systems of silkworm. II. Formation of the juvenile hormone skeletons by farnesyl pyrophosphate synthetase II.

Comparative substrate specificities of farnesyl pyrophosphate synthetases I and II purified from larvae of silkworm, Bombyx mori, were studied by use of the possible biosynthetic intermediates of juvenile hormones in the insect. In the presence of Mn2+ ions farnesyl pyrophosphate synthetase II showed higher activity than synthetase I and the corresponding enzyme from pig liver with the following substrate homologues: (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, (2E,6Z)-3,7-dimethyl-2,6-nonadienyl-, and (2E,6Z)-3-ethyl-7-methyl-2,6-nonadienyl pyrophosphate. When (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, and isopentenyl pyrophosphate were mixed and incubated with farnesyl pyrophosphate synthetase II, (2E,6E,10Z)-3,11-dimethyl-7-ethyl-2,6,10-tridecatrienyl-, (2E,6E,10Z)-3,7,11-trimethyl-2,6,10-tridecatrienyl, and a trace amount of (2E,6E,10Z)-3,7-diethyl-11-methyl-2,6,10-tridecatrienyl pyrophosphate, whose carbon skeletons were the same as those of juvenile hormone I, II, and O, respectively, were formed. (Z)-3-Methyl-2-pentenyl pyrophosphate was produced from 3-ethyl-3-butenyl pyrophosphate as a single product by the action of silkworm isopentenyl pyrophosphate isomerase, though the enzyme activity was much lower with this substrate than with the usual substrate, isopentenyl pyrophosphate.

Animals↗
PubMed1988

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-pinene and (+)- and (-)-camphene.

Cyclase I from Salvia officinalis leaf catalyzes the conversion of geranyl pyrophosphate to the stereo-chemically related bicyclic monoterpenes (+)-alpha-pinene and (+)-camphene and to lesser quantities of monocyclic and acyclic olefins, whereas cyclase II from this plant tissue converts the same acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene and (-)-camphene as well as to lesser amounts of monocyclics and acyclics. These antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H]Linalyl pyrophosphate (3H:14C = 5.14) was tested as a substrate with both cyclases to determine the configuration of the cyclizing intermediate. This substrate with cyclase I yielded alpha-pinene and camphene with 3H:14C ratios of 3.1 and 4.2, respectively, indicating preferential, but not exclusive, utilization of the (3R)-enantiomer. With cyclase II, the doubly labeled substrate gave bicyclic olefins with 3H:14C ratios of from 13 to 20, indicating preferential, but not exclusive, utilization of the (3S)-enantiomer in this case. (3R)- and (3S)-[1Z-3H]linalyl pyrophosphate were separately compared to the achiral precursors [1-3H]geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. With cyclase I, geranyl, neryl, and (3R)-linalyl pyrophosphate gave rise exclusively to (+)-alpha-pinene and (+)-camphene, whereas (3S)-linayl pyrophosphate produced, at relatively low rates, the (-)-isomers. With cyclase II, geranyl, neryl, and (3S)-linalyl pyrophosphate yielded exclusively the (-)-isomer series, whereas (3R)-linalyl pyrophosphate afforded the (+)-isomers at low rates. These results are entirely consistent with the predicted stereochemistries and additionally revealed the unusual ability of these enzymes to catalyze antipodal cyclizations when presented with the unnatural linalyl enantiomer.

Acyclic Monoterpenes↗
PubMed1991

Comparison of indium-111 antimyosin antibody and technetium-99m pyrophosphate localization in reperfused and nonreperfused myocardial infarction.

Recent imaging studies suggest that technetium-99m (Tc-99m) pyrophosphate yields a considerably larger estimate of myocardial infarct size than does indium-111 (In-111) monoclonal antimyosin antibody. To determine whether Tc-99m pyrophosphate may be taken up by reversibly injured myocytes, particularly in the setting of coronary reperfusion, the tissue localization of Tc-99m pyrophosphate and antimyosin antibody was compared in 11 dogs 24 to 68 h after anterior descending coronary artery occlusion (4 dogs with permanent occlusion, 7 with reperfusion). Technetium-99m pyrophosphate and In-111 antimyosin antibody content was determined in serial 2 to 3 mm wide endocardial and epicardial samples taken through the infarct zone in multiple short-axis left ventricular slices. The number of samples with increased In-111 antimyosin antibody (defined as greater than or equal to mean + 2 SD of normal) was not significantly different from that with increased Tc-99m pyrophosphate. This was true in both reperfused and nonreperfused infarcts. However, the intensity of uptake of Tc-99m pyrophosphate exceeded that of In-111 antimyosin antibody, particularly in the border zones of reperfused infarcts, and the area with moderate to marked increase in tracer uptake (greater than or equal to 2 times normal) was significantly larger with Tc-99m pyrophosphate than In-111 antimyosin antibody (p less than 0.001). A specific zone of abnormal Tc-99m pyrophosphate with normal In-111 antimyosin antibody content could not be identified. Histologic evidence of myocardial necrosis was found in virtually every sample with increased In-111 antimyosin antibody, Tc-99m pyrophosphate, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

A model for human calcium pyrophosphate crystal deposition disease: crystallization kinetics in a gelatin matrix.

A model for the deposition of calcium pyrophosphate dihydrate (CPPD) crystals in cartilage observed in human CPPD crystal deposition disease has been developed using diffusion of calcium and pyrophosphate ions through a denatured collagen matrix environment at physiologic pH. This model system uses biological grade gelatin and has allowed for the study of crystal deposition over a wide range of calcium and pyrophosphate concentrations, including physiologic levels. The model has reproducibly formed the two crystallographic dimorphs observed clinically: triclinic and monoclinic calcium pyrophosphate dihydrate. In addition, amorphous calcium pyrophosphate has been identified, and is the first species to form in the crystallization process and transforms to orthorhombic calcium pyrophosphate tetrahydrate. This in turn dissolves with a very localized increase in available pyrophosphate leading to the formation of triclinic and monoclinic calcium pyrophosphate dihydrate. The denatured collagen matrix has allowed for the formation of the two in vivo crystals at pyrophosphate concentrations lower than previously reported in solution studies.

Arthritis↗
PubMed1988

The effect of pyrophosphate on the reaction of myosin with 2,4,6-trinitrobenzene sulphonate.

Myosin was reacted with 2,4,6-trinitrobenzene sulphonate (TNBS) in the presence or absence of Mg-pyrophosphate. The reaction led to trinitrophenylation of lysyl residues which could be divided on the basis of the reaction into three classes: (i) two rapidly reacting lysyl residues (RLR), one residing on each head of myosin, whose rate of reaction depends on the presence of Mg-pyrophosphate; (ii) two lysyl residues which react with intermediate rate (ILR) and reside on the rod segment of myosin; and (iii) the remaining lysyl residues of myosin which react slowly with TNBS. The rate of the trinitrophenylation of RLR was followed spectrophotometrically and enzymatically, measuring an absorbance change at 345 nm, and also changes in K+ (EDTA)-, Mg2+- and Ca2+-activated ATPase activities, respectively. According to analysis of the kinetics of the reaction, Mg-pyrophosphate inhibited the rate of trinitrophenylation in both heads of myosin, not in one head only as was suggested by Miyanishi et al. (J. Biochem Tokyo 85; 1979). Myosin heads (myosin subfragment-1, S-1) were prepared by digesting myosin trinitrophenylated in the absence and presence of Mg-pyrophosphate with chymotrypsin. S-1, with trinitrophenylated RLR, was separated from non-trinitrophenylated S-1 by DEAE cellulose column chromatography. The trinitrophenylated S-1 had a high Mg2+- and a low K+(EDTA)-activated ATPase while the non-trinitrophenylated species had the usual high K+(EDTA)- and low Mg2+-ATPase activity. This results excluded the possibility suggested by Miyanishi et al., that the myosin head, which is resistant to trinitrophenylation in the presence of Mg-pyrophosphate, did not possess K+(EDTA)-activated ATPase activity. The presence of Mg-pyrophosphate during trinitrophenylation substantially affected the enzymic characteristics of the modified myosin. The myosin trinitrophenylated in the presence of Mg-pyrophosphate had a higher K+(EDTA)- and a lower Mg2+-ATPase activity. SH1 (Cys-707) also probably becomes a target of the reaction if myosin is trinitrophenylated in the presence of Mg-pyrophosphate. This is deduced from the following findings: (i) the addition of dithiothreitol after trinitrophenylation partially reversed the loss in the K+(EDTA)-ATPase activity; and (ii) the specific alkylation of the SH1 thiol by 1,5-IAEDANS prior to trinitrophenylation prevented the effect of dithiothreitol on the ATPase activity of myosin. The results indicated that Mg-pyrophosphate induced structural changes in the myosin molecule which influenced the course and possibly the target(s) of trinitrophenylation.

Adenosine Triphosphatases↗