6-thioguanosine-5--triphosphate and 6-thioguanosine-5--diphosphate

Metabolism of thiopurine drugs--azathioprine, 6-mercaptopurine, and 6-thioguanine--has provided a powerful pharmacogenetic model incorporating polymorphism of the enzyme thiopurine methyltransferase (TPMT) and the primary active metabolite, thioguanine nucleotide (TGN). However, a sense of uncertainty about the usefulness of TGNs and other thiopurine metabolites has appeared. This review critically appraises the basis of thiopurine metabolism and reveals the problems and complexities in TGN research. Erythrocyte TGN is used in transplantation medicine and in chronic inflammatory conditions such as Crohn's disease, as a "surrogate" pharmacokinetic parameter for TGN in the target cells: leukocytes or bone marrow. It is not generally appreciated that erythrocytes do not express the enzyme IMP dehydrogenase and cannot convert mercaptopurine to TGN, which explains some of the confusion in interpretation of erythrocyte TGN measurements. TGN routinely measured in erythrocytes derives from hepatic metabolism. Another concern is that TGN are not generally assayed directly: most methods assay the thiopurine bases. Ion-exchange HPLC and enzymatic conversion of TGNs to nucleosides have been used to overcome this, and may reveal undisclosed roles for an unusual cytotoxic nucleotide, thio-inosine triphosphate, and methylated thiopurines. There appear to be additional interactions between xanthine oxidase and TPMT, and folate and TPMT, that could predict leukopenia. Difficult questions remain to be answered, which may be assisted by technological advances. Prospective TGN studies, long overdue, are at last revealing clearer results.

Topics: Azathioprine; Drug Monitoring; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Leukopenia; Mercaptopurine; Methylation; Methyltransferases; Nausea; Thioguanine; Thionucleotides; Xanthine Oxidase

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Topics: Adolescent; Adult; Aged; Azathioprine; Catalysis; Drug Delivery Systems; Enzyme Activation; Erythrocytes; Female; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Male; Middle Aged; Nucleoside-Diphosphate Kinase; Purines; Thionucleotides; Young Adult

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH2PO4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (lambda ex = 325 nm, lambda em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 microliters red blood cells for the three analytes (RSDs < or = 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

Topics: Electrophoresis; Erythrocytes; Fluorescence; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mercaptopurine; Thionucleotides

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.

Topics: Animals; Chromatography, High Pressure Liquid; Enzymes; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mercaptopurine; Phosphorylation; Rabbits; Thionucleotides

Activation of the NADPH oxidase was examined in electrically permeabilized human neutrophils exposed to non-hydrolysable guanine nucleotides. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced a marked increase in the rate of O2 consumption, which was partially resistant to staurosporine, an inhibitor of protein kinase C, under conditions where the response to diacylglycerol was virtually abolished. The respiratory burst elicited by GTP[S] was dependent on the presence of ATP and Mg2+, suggesting involvement of phosphorylation reactions. Accordingly, phosphoprotein formation was greatly stimulated by the guanine nucleotide. The polypeptide phosphorylation pattern induced by GTP[S] was similar to, but not identical with, that observed with diacylglycerol, indicating the activation of kinases other than protein kinase C by the guanine nucleotide. The possible involvement of tyrosine kinases was assessed by immunoblotting using anti-phosphotyrosine antibodies. Treatment of electroporated cells with GTP[S] stimulated the accumulation of tyrosine-phosphorylated proteins. This effect was not induced by diacylglycerol, indicating that tyrosine phosphorylation is not secondary to stimulation of protein kinase C. The results indicate that, in neutrophils, activated G-proteins can stimulate tyrosine kinase and/or inhibit tyrosine phosphatase activity. Changes in the amounts of tyrosine-phosphorylated proteins may signal activation of the respiratory burst.

Topics: Adenosine Triphosphate; Guanine; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Magnesium; Neutrophils; Phosphorylation; Thionucleotides; Tyrosine

The interaction of elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) from Escherichia coli has been investigated by kinetic methods. It was found that EF-Ts purified on an EF-Tu affinity column contained a transphosphorylase activity which could transfer the gamma-phosphate of GTP to [3H]GDP. However, this activity showed different sensitivities to heat and N-ethylmaleimide compared to the EF-Ts activity. Using the chromophoric GDP analogue, 2-amino-6-mercaptopurine riboside 5'-diphosphate (thioGDP), spectrophotometric titrations and stopped-flow experiments enabled the interaction of EF-Tu X thioGDP with EF-Ts and of EF-Tu X EF-Ts with thioGDP to be investigated. The results were analyzed according to the scheme of Chau et al. (Chau, V., Romero, G., and Biltonen, R.L. (1981) J. Biol. Chem. 256, 5591-5596). (Formula: see text) Values for the rate constants obtained were k1 greater than or equal to 2 X 10(8) M-1 s-1, k-1 greater than or equal to 2600 s-1, k2 = 500 s-1, and k-2 = 4 X 10(5) M-1 s-1. The most notable feature of these results is that EF-Ts binds to EF-Tu X thioGDP at a rate approaching that expected for a diffusion-controlled reaction whereas thioGDP binds to EF-Tu X EF-Ts several orders of magnitude more slowly than this. The relevance of these results to the interactions involving GDP is discussed.

Topics: Escherichia coli; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor Tu; Peptide Elongation Factors; Thionucleotides; Tritium

Topics: Azides; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Inosine Monophosphate; Kinetics; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Peptide Elongation Factors; Protein Binding; Ribonucleotides; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Thionucleotides