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Biochemical Pharmacology Jan 19956-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine...
6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.
Topics: Adenine; Adenosine; Antineoplastic Agents; Cell Division; Cell Survival; Cells, Cultured; Guanosine; Humans; Inosine; Mercaptopurine; Methylthioinosine; Nucleosides; Nucleotides; Purine Nucleosides; Ribonucleosides; T-Lymphocytes; Thioinosine; Thionucleotides
PubMed: 7840782
DOI: 10.1016/0006-2952(94)00387-2 -
Molecular Biology of the Cell Jan 1993Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The...
Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.
Topics: 2-Aminopurine; Animals; Cell Line; Methylthioinosine; Myelin Basic Protein; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Nerve Growth Factor; Thioguanine
PubMed: 7680248
DOI: 10.1091/mbc.4.1.71 -
The Journal of Biological Chemistry Jun 1992The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and...
The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.
Topics: Amidophosphoribosyltransferase; Aminoimidazole Carboxamide; Animals; Azaserine; Folic Acid Antagonists; Leukemia, Experimental; Methotrexate; Methylthioinosine; Mice; Purines; Pyrimidines; Ribonucleotides; Tumor Cells, Cultured
PubMed: 1597445
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1992Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal...
Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.
Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.
Topics: Aminopterin; B-Lymphocytes; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Methylthioinosine; Purine Nucleotides; Ribose-Phosphate Pyrophosphokinase
PubMed: 1311306
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1985The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the...
The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.
Topics: Adenosine Diphosphate; Adenosine Kinase; Adenosine Monophosphate; Adenosine Triphosphate; Deoxyadenosines; Female; Humans; Hydrogen-Ion Concentration; Kinetics; Methylthioinosine; Nucleosides; Nucleotides; Phosphorylation; Phosphotransferases; Placenta; Potassium Chloride; Pregnancy; Tubercidin; Vidarabine
PubMed: 2999129
DOI: No ID Found -
The Journal of Biological Chemistry Jun 19845'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted...
5'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted to 2-keto-4-methylthiobutyrate ( ketomethylthiobutyrate ) which is transaminated to methionine. We report that one subline of a heterogeneous human colon carcinoma, DLD-1 Clone D, only forms methylthioribose-1-P from methylthioadenosine or 5'-deoxy-5'-methylthioinosine (methylthioinosine), a deaminated derivative of methylthioadenosine, whereas Clone A converts methylthioadenosine and methylthioinosine to methionine, as shown by growth studies in culture of Clone A and Clone D cells and radioactive studies utilizing [methyl-14C]methylthioadenosine or [methyl-14C]methylthioinosine in the presence of extracts of these cells lines. To characterize this defect, we utilized three protein fractions isolated from rat liver which together convert methylthioribose-1-P to ketomethylthiobutyrate . Addition of only Fraction A to Clone D sonicates restores its ability to convert methylthioadenosine to methionine. This fraction is responsible for converting methylthioribose-1-P to 5- methylthioribulose -1-phosphate; radioactive studies confirm this observation. Thus, Clone D is deficient in an enzyme contained in Fraction A; this represents a qualitative biochemical difference between the two clones derived from a single human tumor.
Topics: Adenosine; Animals; Cell Line; Colonic Neoplasms; Deoxyadenosines; Humans; Liver; Methionine; Rats; Ribosemonophosphates; Ribulosephosphates; Thioglycosides; Thionucleosides
PubMed: 6725268
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1984Methylthioinosine (MeSno) is a purine nucleoside analog which is cytotoxic to a number of cultured cell lines including the Reuber H35 hepatoma cells used in the present...
Methylthioinosine (MeSno) is a purine nucleoside analog which is cytotoxic to a number of cultured cell lines including the Reuber H35 hepatoma cells used in the present studies. It has also been observed to cause a rapid profound loss of tyrosine aminotransferase activity in H35 cells well before the onset of any measurable cytotoxicity. The effect is both time and concentration dependent. MeSno does not acutely inhibit synthesis of the enzyme as evidenced by the ability of glucocorticoids or cAMP analogs to induce the enzyme to the same extent in the presence or absence of the drug. The enzyme in extracts of cells treated with the drug is essentially identical with the enzyme from extracts of control cells in terms of thermal stability, immunoprecipitability, and affinities for substrates and cofactor. Addition of MeSno to cell extracts and mixing experiments suggests that the thiopurine does not have any direct effect on enzyme activity. Immunochemical analysis of the rates of synthesis and degradation of the aminotransferase have shown that the enzyme is degraded approximately 3-4 times more rapidly in cells treated with the drug than in control cells. At the same time there is no inhibition of the rate of synthesis of the enzyme.
Topics: Animals; Dexamethasone; Dose-Response Relationship, Drug; Hot Temperature; Inosine; Liver Neoplasms, Experimental; Methylthioinosine; Rats; Tyrosine Transaminase
PubMed: 6141163
DOI: No ID Found -
The Journal of Biological Chemistry Feb 1981
Topics: Adenine; Cell Division; Feedback; Fibroblasts; Humans; Hypoxanthines; Inosine; Kinetics; Methylthioinosine; Phosphotransferases; Ribose-Phosphate Pyrophosphokinase; Skin
PubMed: 6257688
DOI: No ID Found -
European Journal of Biochemistry Feb 19815'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and...
5'-Methylthioadenosine nucleosidase (EC 3.2.2.9), the enzyme which catalyzes hydrolytic cleavage of 5'-methylthioadenosine with the formation of adenine and 5'-methylthioribose, has been purified to homogeneity from Lupinus luteus seeds. The nucleosidase has a native molecular weight of 62 000 and consists of two identical subunits, as judged by gel filtration and dodecylsulfate/polyacrylamide gel electrophoresis. The nucleosidase exhibits highest specificity towards the natural substrate with a Km of 4.1 X 10(-7) M for 5'-methylthioadenosine. It does not cleave adenine from S-adenosylhomocysteine. Among the synthetic analogs of 5'-methylthioadenosine tested, eleven compounds appear to be able to substitute as substrates. Furthermore, the enzyme can liberate hypoxanthinine from six inosyl (deaminated) derivatives obtained by enzymatic deamination of 5'-methylthioadenosine and its synthetic analogs. The Km for 5'-methylthioinosine is 55 microM, and the maximal velocity about 50-times lower than for 5'-methylthioadenosine. The reaction catalyzed by the nucleosidase can be inhibited by adenine (Ki = 11 microM), 3-deazaadenine (Ki = 19 microM), and 9-erythro(2-hydroxyl-3-nonyl)adenine (ki = 37 microM).
Topics: Adenosine; Kinetics; Molecular Weight; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Seeds; Substrate Specificity; Thionucleosides
PubMed: 6783408
DOI: 10.1111/j.1432-1033.1981.tb05148.x -
European Journal of Biochemistry Nov 1979The occurrence of 5'-methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87 degrees C, is reported. It represents...
The occurrence of 5'-methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87 degrees C, is reported. It represents the first example in prokaryotes of a phosphoryolytic cleavage of the thioether. The reaction products, purified by ion-exchange chromatography, have been identified as 5-methylthioribose-1-phosphate and adenine by several analytical procedures. The enzyme has been purified to homogeneity in 32% yield by using DEAE-cellulose and hydroxyapatite chromatography, gel filtration and isoelectric focusing. The enzyme shows a high degree of thermophilicity, its temperature optimum being at 93 degrees C; furthermore no loss of activity is observable after exposure for 1 h at 100 degrees C. The kinetic data indicate a sequential mechanism of the reaction. The apparent Km values are 0.095 mM for 5'-methylthioadenosine and 6.1 mM for phosphate. The specificity of the reaction is rather strict. Experiments performed with analogues of the substrate, i.e. 5'-methylthioinosine, 5'-dimethylthioadenosine sulfonium salt, 5'-n-butylthioadenosine, 5'-isobutylthioadenosine, 5'-isobutylthioinosine, adenosylhomocysteine, 5'-thioethanoladenosine, adenosine, indicate the relevance of the adenine amino group and the sulfur in thioether form in the binding to the enzyme protein.
Topics: Adenosine; Bacteria; Drug Stability; Hot Temperature; Kinetics; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides
PubMed: 118001
DOI: 10.1111/j.1432-1033.1979.tb19723.x