Decreased methionine synthesis in purine nucleoside-treated T and B lymphoblasts and reversal by homocysteine.

Boss, Gerry R.; Pilz, Renate B.

doi:10.1172/jci111536

Cited by 11 publications

(7 citation statements)

References 29 publications

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“…Origin and culture of lymphoblasts CCRF-CEM and MOLT-4 were derived from acute human T cell leukaemias; WIL2 is a splenic-derived human B lymphoblastoid cell line [12]. MGL8B2, 889,…”

Section: Methodsmentioning

confidence: 99%

“…AdoHcy contribute the carbon atoms to positions 8 and 2 of the purine ring, respectively. 679 and MGL28 were established by Epstein-Barr virus transformation of peripheral blood B lymphocytes from a normal, an adenosine deaminase-deficient, a purine nucleoside phosphorylase-deficient and a methylene tetrahydrofolate reductase-deficient individual, respectively [12,14]. The adenosine deaminase-deficient and purine nucleoside phosphorylase-deficient cells had < 1 % of residual enzyme activities as compared with cells derived from healthy controls, whereas the methylene tetrahydrofolate reductase-deficient cells had approx.…”

Section: Vol 242mentioning

confidence: 99%

“…The adenosine deaminase-deficient and purine nucleoside phosphorylase-deficient cells had < 1 % of residual enzyme activities as compared with cells derived from healthy controls, whereas the methylene tetrahydrofolate reductase-deficient cells had approx. 18% of the normal enzyme activity [12,14]. Cells were routinely cultured in RPMI-1640 medium (Irvine Scientific, Irvine, CA, U.S.A.) supplemented with 10% (v/v) fetal bovine serum as previously described [14].…”

Section: Vol 242mentioning

confidence: 99%

“…We showed recently that purine deoxynucleosides decrease methionine synthesis from homocysteine in cultured human T lymphoblasts and, less severely, in B lymphoblasts [12]. This defect could be reversed by the addition of homocysteine, suggesting that the purine deoxynucleosides caused trapping of 5-methyltetrahydrofolate.…”

Section: Introductionmentioning

confidence: 97%

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Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts

Boss¹

1987

Biochemical Journal

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Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.

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“…Origin and culture of lymphoblasts CCRF-CEM and MOLT-4 were derived from acute human T cell leukaemias; WIL2 is a splenic-derived human B lymphoblastoid cell line [12]. MGL8B2, 889,…”

Section: Methodsmentioning

confidence: 99%

Section: Vol 242mentioning

confidence: 99%

Section: Vol 242mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

See 2 more Smart Citations

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts

Boss¹

1987

Biochemical Journal

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“…Radioactivity on the filters was quantitated by liquid scintillation counting, and the assay was linear with time from 1 to 3 h and with cell number from 2 to 5 ϫ 10 6 . Measurement of Rates of Methionine and Serine Synthesis-Rates of methionine and serine synthesis were measured as described previously following [ 14 C]formate incorporation into methionine and serine in protein (22)(23)(24) (Fig. 1).…”

Section: Measurement Of [ 14 C]methyl-tetrahydrofolate Incorporation mentioning

confidence: 99%

Nitric Oxide Inhibits Methionine Synthase Activity in Vivo and Disrupts Carbon Flow through the Folate Pathway

Danishpajooh

Gudi

Chen

et al. 2001

Journal of Biological Chemistry

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Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B 12 ) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [ 14 C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [ 14 C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.Vitamin B 12 deficiency leads to pernicious anemia and subacute combined degeneration of the spinal cord (1). Pernicious anemia is characterized by megaloblastic erythropoiesis and is secondary to decreased activity of methionine synthase, one of two mammalian enzymes that requires vitamin B 12 (cobalamin) as a cofactor (2, 3). Methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine via a methylcobalamin intermediate with cycling of cobalamin between the ϩ1 valency state (i.e. cbl(I)) 1 and the ϩ3 valency state (i.e. cbl(III)) (2, 3). Methyltetrahydrofolate is the major intracellular storage form of folates, and its synthesis from 5,10-methylene tetrahydrofolate is essentially irreversible in vivo (2, 4) (Fig. 1). Thus, decreased methionine synthase activity leads to trapping of intracellular folates as 5-methyltetrahydrofolate, and the megaloblastic anemia of vitamin B 12 deficiency is virtually indistinguishable from the megaloblastosis of folate deficiency (1).Nitric oxide (NO) is produced by most cell types and regulates a diverse array of biological functions (5, 6). NO has been reported to inhibit methionine synthase activity in vitro (7-9), and it might be expected to bind to the cobalt in cobalamin because (i) NO binds tightly to the iron in heme (10); (ii) ferrous heme and cbl(III) are isoelectronic; and (iii) in both heme and cobalamin, the metal ion is coordinated to four in-plane nitrogen atoms of a tetrapyrrole ring and has two out-of-plane ligands (3...

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Differential deoxyadenosine toxicity to immature rabbit cartilage in vitro. a model for the chondro‐osseous dysplasia of adenosine deaminase deficiency

Wortmann

Veum

Ryan

et al. 1989

Arthritis & Rheumatism

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Deoxyadenosine metabolism was investigated in rabbit growth plate and articular cartilage to elucidate the biochemical basis for the chondro-osseous dysplasia observed in adenosine deaminase (ADA) deficiency. Models of ADA deficiency, the combination of deoxyadenosine and either of 2 ADA inhibitors, were selectively toxic to immature cartilage, supporting the hypothesis that the chondro-osseous dysplasia of ADA deficiency is the consequence of the enzyme deficiency. Depletion of ATP may play a role in the altered chondrocyte viability and function observed in this model.Adenosine deaminase (ADA) (EC 3.5.4.4) catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. An inherited deficiency of ADA causes an autosomal recessive form of severe combined immunodeficiency disease (1-3). Clinically, ADA-deficient individuals suffer from overwhelming infection and malabsorption. In addition to immune dysfunction, ADA-deficient individuals develop abnormalities in bone and cartilage, termed chondro-osseous dysplasia ( 4 6 ) , which is recognized by characteristic radiographic changes and is associated with a distinctive histopathologic pattern. Although the radiographic changes are not entirely specific, they are quite prevalent in chondro-osseous dysplasia and improve with successful enzyme replacement therapy (7,s).The absence of ADA activity results in the predictable increased concentrations of deoxyadenosine and adenosine in deficient individuals (9,lO). In vivo, the accumulation of deoxyadenosine leads to increased concentrations of deoxyATP in erythrocytes, peripheral blood lymphocytes, and bone marrow cells (10,ll). Precisely how the accumulation of these metabolites causes immune dysfunction is uncertain. Possibilities include inhibition of ribonucleotide reductase (EC 1.17.4.1) by deoxyATP (10,l I), inhibition and inactivation of S-adenosyl-L-homocysteine (SAH) hydrolase (EC 3.3.1.1) by adenosine and deoxyadenosine (12,131, and ATP depletion (1416). Also, deoxyadenosine and its metabolites have been observed to cause accumulation of DNA strand breaks (17,18), inhibit RNA synthesis (18,19), lower NAD pools (lS), and inhibit methionine synthesis (20).The metabolic abnormalities that cause the chondro-osseous dysplasia are also not understood. We have investigated the effects of ADA inhibition in vitro on chondrocyte viability and function. Our results support the hypothesis that the chondro-osseous dysplasia observed in ADA deficiency is the consequence of the disordered purine metabolism that results when ADA activity is absent, and suggest that ATP depletion may play a significant role in the pathogenesis of the cartilage and bone pathology observed.

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Decreased methionine synthesis in purine nucleoside-treated T and B lymphoblasts and reversal by homocysteine.

Cited by 11 publications

References 29 publications

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts

Nitric Oxide Inhibits Methionine Synthase Activity in Vivo and Disrupts Carbon Flow through the Folate Pathway

Differential deoxyadenosine toxicity to immature rabbit cartilage in vitro. a model for the chondro‐osseous dysplasia of adenosine deaminase deficiency

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