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At least 19 recordsLinked to original sources
PubMed1989

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs.

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.

Animals↗
PubMed1979

The enzymatic hydrolysis of the phosphate ester bond in some thionucleotides.

We prepared the 5'- and 3'-O-phosphorothioate esters of the antitumor agent O2 : 2'-anhydro-1-beta-D-arabinosylcytosine. We also included in this study esters of 2'-thio-2'-deoxycytidine, namely, 2'-S-dCyd-2' : 3'-P, 2'-S-dCyd-2'-P, and 2'-S-dCyd-3'-P, along with natural nucleotides. These compounds were subjected to the action of Escherichia coli alkaline phosphatase, potato acid phosphatase, and bovine pancreatic ribonuclease A. The data were analyzed by Lineweaver-Burk plots to obtain Km and KI values. Only 2'-S-dCyd-2'-P was a substrate for alkaline phosphatase; the anhydro-araCyt phosphorothioates were good competitive inhibitors, while 2'-S-dCyd-3'-P did not associate with the enzyme. Acid phosphatase hydrolyzed all four monoesters investigated, including the S-phosphorothioate. The cyclic phosphorothioate, 2'-S-dCyd-2' : 3'-P was neither hydrolyzed by, nor associated with, ribonuclease A. ORD spectroscopy was also used in an attempt to relate the structural features of analogs to the peculiarity of their hydrolysis.

Acid Phosphatase↗
PubMed1984

Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP.

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.

Adenine Nucleotides↗
PubMed1968

Presence and function of sulur-containing transfer ribonucleic acid of Bacillus subtilis.

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.

Bacillus subtilis↗
PubMed1993

Desulfuration of 6-mercaptopurine. The basis for the paradoxical cytotoxicity of thiopurines in cultured human leukemic cells.

The thiopurines have a wide array of effects on purine metabolism, but the primary mechanism of cytotoxicity for both 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) appears to be incorporation of drug into DNA following conversion to the thioguanylate form. In murine leukemic cell lines exposed to a range of thiopurine concentrations in vitro, cell survival curves have displayed a phenomenon termed paradoxical cytotoxicity, defined as a decrease in cytotoxicity with increasing drug concentration. The paradoxical cytotoxicity of thiopurines has usually been attributed to concentration-dependent perturbations in the cell cycle. The present study assessed whether the paradoxical cytotoxicity of 6-MP occurred in cultured human leukemic cells, and investigated the biochemical and cell-cycle alterations occurring in these lines at thiopurine concentrations associated with the reverse of cytotoxicity. Paradoxical cytotoxicity was observed in the two human leukemic cell lines examined, but only when 6-MP concentrations exceeded 100 microM. The extent of incorporation of 6-MP metabolites into DNA as thiol-versus non-thiol-containing metabolites was analyzed by performing parallel experiments with 14C- and 35S-radiolabeled drug. With 5 microM 6-MP, approximately 50% of drug was incorporated into DNA as a thionucleotide; however, with increasing drug concentrations, the degree of thionucleotide incorporation remained unchanged or decreased, and the amount incorporated as the desulfurated metabolite (presumably adenylate or guanylate) increased. With 500 microM 6-MP, less than 10% of the drug was incorporated as the thionucleotide. Perturbations in cell cycle reflected the relative amounts of thiol- and non-thiol-containing nucleotide formed at various concentrations of 6-MP. These results suggest that thiopurines may be vulnerable to a unique mechanism of detoxification, in which a human cell can metabolize a cytotoxic drug to a comparatively potent "self-rescue" agent.

Carbon Radioisotopes↗
PubMed1980

31P NMR quantitation of the displacement of equilibria of arginine, creatine, pyruvate, and 3-P-glycerate kinase reactions by substitution of sulfur for oxygen in the beta phosphate of ATP.

31P NMR measurements have been found to be a convenient means for simultaneously measuring the concentrations of several species in the equilibrium mixtures of the reactions catalyzed by arginine kinase and creatine kinase. MgATP + X in equilibrium MgADP + XP where X = arginine or creatine and XP = P-arginine or P-creatine. The free energy of phosphorylaton of various metabolites by adenosine 5'-O-(2-thiotriphosphate) at pH 8.0 and 30 degrees C is more exergonic than the corresponding phosphorylations by ATP by about 2.5 kcal/mol, resulting in a displacement of the equilibrium toward the nucleoside diphosphates by a factor of approximately 60. Since this factor does not depend on the nature of the metabolite, the equilibrium constants of thionucleotide reactions may be used to determined the equilibrium constants of corresponding oxynucleotide reactions which lie too far toward ATP. The equilibrium constants of the oxynucleotide reactions catalyzed by pyruvate kinase and 3-P-glycerate kinase calculated by this method from the experimentally determined equilibrium constants of the corresponding thionucleotide reactions are 3.1 x 10(-4) and 2.9 x 10(-4), respectively, under the experimental conditions used. The equilibrium constants and degree of stereoselectivity of the arginine kinase reaction are altered when Ca2+ replaces Mg2+ as the activating metal ion.

Adenosine Triphosphate↗
PubMed1999

Metabolism of 6-mercaptopurine in the erythrocytes, liver, and kidney of rats during multiple-dose regimens.

PURPOSE: To describe the metabolism of 6-mercaptopurine (6-MP) in erythrocytes and tissues of rats after repeated administration of 6-MP at two dose levels and to provide evidence that in vivo modulation of 6-MP anabolism can be obtained by simultaneous treatment with ribavirin or hydroxyurea, two inhibitors of enzymes involved in the bioactivation of 6-MP to the active 6-thioguanine nucleotides (6-TGN). METHODS: Rats were treated i.p. with 6-MP at 12.5 and 25 mg/kg daily for 12 days and erythrocyte, liver, and kidney levels of 6-mercaptopurine nucleotides (6-MPN) and 6-TGN were investigated during the accumulation phase and for 50 days after the end of treatment. In combination studies, ribavirin at 75 and 100 mg/kg per day (for 6-MP, 25 and 12.5 mg/kg per day) or hydroxyurea at 200 mg/kg per day were given i.p. for 12 days. The measurements of thionucleotide levels in rat samples were performed by high-pressure liquid chromatography (HPLC). RESULTS: The maximal concentration (Cmax) and the area under the concentration versus time curve (AUC) of 6-MPN and 6-TGN in erythrocytes and tissues increased significantly after the administration of 6-MP at 25 mg/kg per day as compared with 12.5 mg/kg per day. In particular, the Cmax and AUC of 6-TGN in erythrocytes of rats treated with 6-MP at 25 mg/kg per day were approximately 5-fold higher than the 6-TGN values observed following treatment at 12.5 mg/kg per day. Moreover, 6-TGN levels in erythrocytes were significantly higher than those of 6-MPN (910.9+/-53.1 and 286.8+/-23.4 pmol/8 x 10(8) cells for 6-TGN and 6-MPN, respectively, P < 0.05) after treatment with 6-MP at 25 mg/kg per day. The administration of ribavirin, an inhibitor of inosine monophosphate dehydrogenase, in association with 6-MP increased the amount of 6-MPN detected in erythrocytes and tissues while reducing 6-TGN levels in samples. The production and accumulation of 6-MPN and 6-TGN were increased in erythrocytes and tissues by hydroxyurea, an inhibitor of ribonucleotide reductase. Finally, a significant correlation between thionucleotide concentrations and erythrocyte counts was observed. CONCLUSION: The overall results demonstrate that 6-MP is actively metabolized in rats and that its biotransformation can be modulated by agents acting on enzymes of the purine metabolism, resulting in significant changes in erythrocyte and tissue levels of 6-MPN and 6-TGN. These findings provide evidence that the rat is a suitable model for investigation of the metabolism of 6-MP and its possible pharmacologic modulation.

Animals↗
PubMed1989

A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase.

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.

Escherichia coli↗
PubMed1987

Assay of 6-thioinosinic acid and 6-thioguanine nucleotides, active metabolites of 6-mercaptopurine, in human red blood cells.

A highly sensitive reversed-phase high-performance liquid chromatographic assay, with ultraviolet detection, for 6-thioinosinic acid and the 6-thioguanine nucleotides (6TGNs) was developed. The 6TGNs are major red blood cell metabolites of the immunosuppressive agent azathioprine and the cytotoxic drugs 6-thioguanine and 6-mercaptopurine. The assay is based on the specific extraction, via phenyl mercury adduct formation, of the thiopurine released on acid hydrolysis of the thionucleotide metabolite. Red blood cell 6TGN concentrations in eighteen leukaemic children receiving chronic 6-mercaptopurine chemotherapy were measured and compared to a previously published spectrophotofluorometric assay. Linear regression analysis gave r = 0.991; P less than 0.001; y = 40 + 0.94x.

Chromatography, High Pressure Liquid↗
PubMed1998

Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

Animals↗
PubMed1988

Catalytic mechanism of biotin carboxylase: steady-state kinetic investigations.

Biotin carboxylase was purified from Escherichia coli by a new procedure, and its steady-state kinetic parameters were examined. MgATP and bicarbonate add to the enzyme randomly, followed by addition of biotin. Both bicarbonate and MgATP add in rapid equilibrium. A catalytic base with a pK of 6.6 is observed in V/K profiles. Inactivation studies also revealed a sulfhydryl group in the active site that is essential for catalysis. It is proposed that the acid-base catalysts are necessary for the tautomerization of biotin, which presumably enhances its nucleophilicity toward the carboxyl group donor. A second enzymic group with a pK of 6.6, whose role is unknown, is seen in Vmax profiles. The pH profiles for the biotin carboxylase catalyzed phosphorylation of ADP by carbamoyl phosphate have the same shape as the profiles for the forward reaction, which demonstrates that the enzymic bases assume the same protonation states for catalysis of transphosphorylation in either direction. The lack of reactivity of thionucleotide analogues of ATP when Mg is used as the divalent metal ion suggests that both metal ions required for reaction coordinate to the nucleotide. The second metal ion appears to be absolutely required for reaction and not merely an activator of the reaction. Characterization of a bicabonate-dependent biotin-independent ATPase activity strongly suggests that carboxylation proceeds via a carboxyphosphate intermediate.

Adenosine Triphosphatases↗
PubMed1988

Stereoselectivity of the guanyl-exchangeable nucleotide-binding site of tubulin probed by guanosine 5'-O-(2-thiotriphosphate) diastereoisomers.

The active site of the exchangeable nucleotide-binding site of tubulin was studied by using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) where the phosphorus atom to which sulfur is attached is chiral. Turbidimetric measurements were used to follow kinetics, and electron microscopy was used to evaluate polymeric forms. Both isomers at 0.5 mM promoted the assembly of tubulin in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid, 30% glycerol, 3 mM MgCl2, and 1 mM EGTA, pH 6.6, 23-37 degrees C. GTP beta S(A) promoted assembly into microtubules, although a few bundles were also found by electron microscopy. However, GTP beta S(B) induced assembly of tubulin into bundles of sheets and microtubules. As expected, 0.5 mM GTP induced tubulin to assemble into microtubules, thin sheets, and a few bundles. Both GTP and GTP beta S(A) were hydrolyzed in the tubulin polymers. However, more than 95% of the bound GTP beta S(B) was not hydrolyzed. Higher concentrations of GTP beta S(B), i.e., 1 mM, also induced bundles of sheets and microtubules, with 86% of the thionucleotide bound as the triphosphate. The GTP beta S(B)-induced polymers were considerably more cold stable than the GTB beta S(A)-induced microtubules, which were more cold stable than GTP-induced polymers. Mg(II) (2-5 mM) had minimal effects on the structures induced by GTP beta S(A) or -(B) isomers in the tubulin assembly system. However, at 1 mM Mg(II), no assembly was found with GTP beta S(A) and tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Exploiting nucleotide thiophosphates to probe mechanistic aspects of Escherichia coli DNA gyrase.

The interaction of DNA gyrase with ATP has been probed using a range of thiophosphate ATP analogs. ATP gammaS is not detectably hydrolyzed by gyrase but can support limited, probably catalytic, DNA supercoiling. ATP gammaS is a good inhibitor of both ATP hydrolysis and ATP-supported supercoiling. In contrast, both ATP alphaS(Rp) and ATP betaS(Rp) have been shown to be good substrates for the ATPase reaction of gyrase and to support catalytic DNA supercoiling. The corresponding Sp diastereoisomers do not support significant levels of supercoiling and are not readily hydrolyzed, but are shown to be reasonable inhibitors of gyrase. For ATP alphaS(Rp), the supercoiling and ATPase activities appear to be tightly coupled with the thionucleotide being apparently a better substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis. In the case of ATP betaS(Rp), DNA supercoiling and nucleotide hydrolysis appear to be uncoupled in that ATP betaS(Rp) is almost as good a substrate as ATP for the ATPase reaction of both intact gyrase and the 43 kDa GyrB fragment, whereas it only supports slow DNA supercoiling; the mechanistic consequences of these observations are discussed in terms of a new model for energy coupling in gyrase. DNA gyrase has been shown to be capable of catalyzing DNA supercoiling in the presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2+, or Cd2+. The pronounced diastereoselectivity seen in both the DNA supercoiling and ATPase activity with ATP alphaS and ATP betaS together with evidence from the X-ray structure of the 43 kDa GyrB-ADPNP-Mg complex is consistent with metal ion coordination at both of these sites, and probably to the gamma-phosphoryl center during turnover. Thus, the absolute configuration of the catalytically active Mg2+-ATP complex is likely to involve coordination to the pro-S oxygens at both P alpha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP complex with the lambda-exo configuration.

Adenosine Triphosphatases↗
PubMed1994

Differential cDNA cloning by enzymatic degrading subtraction (EDS).

We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate the utility of EDS by constructing a subtractive library enriched for cDNAs expressed in adult but not in embryonic rat brains.

Animals↗