Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Diphosphates”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed2001

Artificial substrates of medium-chain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases.

We examined the reactivity of 3-alkyl group homologues of farnesyl diphosphate or isopentenyl diphosphate for medium-chain prenyl diphosphate synthases, hexaprenyl diphosphate- or heptaprenyl diphosphate synthase. But-3-enyl diphosphate, which lacks the methyl group at the 3-position of isopentenyl diphosphate, condensed only once with farnesyl diphosphate to give E-norgeranylgeranyl diphosphate by the action of either enzyme. However, norfarnesyl diphosphate was never accepted as an allylic substrate at all. 3-Ethylbut-3-enyl diphosphate also reacted with farnesyl diphosphate giving a mixture of (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all-E)-3,7-diethyl-11,15,19-trimethylicosa-2,6,10,14,18-pentaenyl diphosphates by hexaprenyl diphosphate synthase. On the other hand, heptaprenyl diphosphate synthase reaction of 3-ethylbut-3-enyl diphosphate with farnesyl diphosphate gave only (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate.

Alkyl and Aryl Transferases↗
PubMed2001

Mechanism of monoterpene cyclization: stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (-)-limonene synthase from spearmint and (-)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.

Binding Sites↗
PubMed2001

Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

Alkyl and Aryl Transferases↗
PubMed2004

Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis.

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when omega,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the alpha-isoprene unit in the Z configuration, indicating that Rv1086 (omega,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.

Alkyl and Aryl Transferases↗
PubMed2002

Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization.

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.

Abies↗
PubMed1991

Formation of Z,E,E-geranylgeranyl diphosphate by rat liver microsomes.

Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.

Alkyl and Aryl Transferases↗
PubMed2003

Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis.

Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.

Alkyl and Aryl Transferases↗
PubMed1999

Protein design of geranyl diphosphate synthase. Structural features that define the product specificities of prenyltransferases.

Geranyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give a C(10) compound, geranyl diphosphate, which is a precursor of all monoterpenoids. However, the gene has not been isolated from any organisms. To examine the possibility that geranyl diphosphate synthase has evolved from a common ancestor of the prenyltransferase family and to predict the active site structure, we tried to convert Bacillus stearothermophilus farnesyl diphosphate synthase to geranyl diphosphate synthase, according to our previous findings. Several mutated farnesyl diphosphate synthases that have single amino acid substitutions before the first aspartate-rich motif were constructed. A mutated enzyme that has the replacement of serine by phenylalanine at the fourth position before the motif exclusively produced geranyl diphosphate when dimethylallyl diphosphate was used as the primer, and hardly accepted geranyl diphosphate as a primer, indicating that this mutation causes the conversion to geranyl diphosphate synthase. This result supports the idea that the product specificities of all members of the E-prenyltransferase family are mainly defined by a few structural features: the amino acids at the fourth position and the fifth position before the first aspartate-rich motif, and the insertion of two amino acids in the motif. This suggests that natural geranyl diphosphate synthases might have an active site structure similar to that of the mutated enzyme.

Amino Acid Sequence↗
PubMed1993

Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.

When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.

Animals↗
PubMed1993

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures.

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.

Journal Article↗
PubMed2003

Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase.

trans -Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate to produce the C(30)-C(50) prenyl diphosphates, which are precursors of the side chains of prenylquinones. Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans -prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans -long-chain prenyl diphosphate synthases from Arabidopsis thaliana. The cDNA was heterologously expressed in Escherichia coli, and the recombinant His(6)-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C(45)) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C(15)) and geranylgeranyl diphosphate (GGPP; C(20)), but did not accept either the C(5) or the C(10) allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73 microM and 1.61 microM respectively. We also performed an analysis of the side chains of prenylquinones extracted from the A. thaliana plant, and showed that its major prenylquinones, i.e. plastoquinone and ubiquinone, contain the C(45) prenyl moiety. This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains. This is the first established trans -long-chain prenyl diphosphate synthase from a multicellular organism.

Alkyl and Aryl Transferases↗
PubMed2000

Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus.

A member of the medium-chain prenyl diphosphate synthases, Bacillus stearothermophilus heptaprenyl diphosphate synthase, catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce (all-E)-C35 prenyl diphosphate as the ultimate product. We previously showed that the product specificity of short-chain prenyl diphosphate synthases is regulated by the structure around the first aspartate-rich motif (FARM). The FARM is also conserved in a subunit of heptaprenyl diphosphate synthase, component II', which suggests that the structure around the FARM of component II' regulates the elongation. To determine whether component II' regulates the product chain length by a mode similar to that of the short-chain prenyl diphosphate synthases, we replaced a bulky amino acid at the eighth position before the FARM of component II', isoleucine 76, by glycine and analyzed the product specificity. The mutated enzyme, I76G, can catalyze condensations of isopentenyl diphosphate beyond the native chain length of C35. Moreover, two mutated enzymes of A79Y and S80F, which have a single replacement to the aromatic residue at the fourth or the fifth position before the FARM, mainly yielded a C20 product. These results strongly suggest that a common mechanism controls the product chain length of both short-chain and medium-chain prenyl diphosphate synthases and that, in wild-type heptaprenyl diphosphate synthase, the prenyl chain can grow on the surface of the small residues at positions 79 and 80, and the elongation is precisely blocked at the length of C35 by isoleucine 76.

Alkyl and Aryl Transferases↗
PubMed1996

Conversion of product specificity of archaebacterial geranylgeranyl-diphosphate synthase. Identification of essential amino acid residues for chain length determination of prenyltransferase reaction.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from approximately 1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T.(1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.

Alcohols↗
PubMed1976

The metal ion catalyzed decomposition of nucleoside diphosphate sugars.

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.

Cations, Divalent↗
PubMed1994

Hexose diphosphates and phosphofructokinase in rat brain during development.

Fructose 2,6-diphosphate and glucose 1,6-diphosphate concentrations were determined during late gestation and over the course of suckling in rat brain cortex and cerebellum. Cortex fructose 2,6-diphosphate concentration was greatest in neonatal animals and gradually declined thereafter by 25% to reach the adult level at 15 days of age. In contrast, the glucose 1,6-diphosphate concentration increased 4-fold over the same period to reach its highest level by postnatal day 15. Neither cerebellar fructose 2,6-diphosphate nor glucose 1,6-diphosphate concentrations varied significantly. Six day cortex 6-phosphofructo-1-kinase was less sensitive to inhibition by citrate than the enzyme obtained from 15 day pups, and fructose 2,6-diphosphate was better than glucose 1,6-diphosphate at relieving the inhibition imposed by citrate at either age. It is suggested that the rise in cerebral glucose use which occurs during suckling cannot be attributed to either changes in the concentrations of fructose 2,6-diphosphate or glucose 1,6-diphosphate, or the age-related differential sensitivity of 6-phosphofructo-1-kinase toward these effectors.

Animals↗
PubMed1990

Fructose 1-6 diphosphate prevents intestinal ischemic reperfusion injury and death in rats.

This study of ischemic and postischemic reperfusion intestinal injury in rats evaluates the potential therapeutic value of fructose 1-6 diphosphate on the basis of its ability to enhance anaerobic carbohydrate metabolism during ischemia and to prevent additional tissue injury after reestablishing blood flow by inhibiting the neutrophils to produce oxygen free radicals. In pursuit of this goal, 28 rats were randomized into 4 groups: pretreated with fructose 1-6 diphosphate (n = 7); pretreated with glucose (n = 7); post-reperfusion treated with fructose 1-6 diphosphate (n = 7); and post-reperfusion treated with saline (n = 7). Five additional rats were sham operated. Following 30 min occlusion of the superior mesenteric artery, all rats received their respective treatments for 5 days. Post-reperfusion arterial pressure was significantly lower in the control rats (p less than 0.001) as well as when compared with the fructose 1-6 diphosphate groups (p less than 0.001). Significant increase in white blood cell counts occurred in the controls (p less than 0.001), whereas in the fructose 1-6 diphosphate groups white blood cell counts were no different from preischemic values. All control rats that died in less than 5 days had transmural intestinal necrosis, whereas in 3 of the controls that survived 5 days, partial intestinal necrosis was noted. Only one fructose 1-6 diphosphate-treated rat had partial intestinal necrosis. The overall 5-day survival was 100% for sham-operated rats, 93% for fructose 1-6 diphosphate-treated rats, and 21% for controls (fructose 1-6 diphosphate vs. controls, p less than 0.001; fructose 1-6 diphosphate vs. sham, NS). The results are discussed and explained in terms of the postulated mechanism based on the pharmacological properties of fructose 1-6 diphosphate.

Animals↗
PubMed1993

Effect of site-directed mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity.

All polyprenyl synthases catalyze the condensation of the allylic substrate, isopentenyl diphosphate, with a specific homoallylic diphosphate substrate. Polyprenyl synthases from Homo sapiens, Ratus rattus, Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and Erwinia herbicola contain two conserved "aspartate-rich domains" (Ashby, M.N., and Edwards, P.A. (1992) J. Biol. Chem. 267, 4128-4136). In order to determine the importance of these domains in catalysis, the conserved aspartates or arginines in domains I and II of rat farnesyl diphosphate synthase were individually mutated to glutamate or lysine, respectively. The putative "active site" arginine (Brems, D.N., Breunger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711-3718) was mutated to lysine. Each mutant enzyme was overexpressed in E. coli and purified to apparent homogeneity. Detailed kinetic analyses of the wild type and mutant enzymes indicated that mutagenesis of Asp104, Asp107, Arg112, Arg113, and Asp243 resulted in a decreased Vmax of approximately 1000-fold compared to wild type. However, no significant change in the Km values for either the isopentenyl diphosphate or geranyl diphosphate substrate were observed. The results strongly suggest that these amino acids, and to a lesser extent Asp244, are involved in either the condensation of isopentenyl diphosphate and geranyl diphosphate to form farnesyl diphosphate and/or the release of the farnesyl diphosphate product from farnesyl diphosphate synthase. The conservation of these amino acid residues in different enzymes from several species suggests that these domains play a similar role in other polyprenyl synthases.

Alkyl and Aryl Transferases↗
PubMed1979

Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.

Adenosine Diphosphate↗