Studies on Normal and Leukemic Leukocytes. Iv. Tetrahydrofolate-Dependent Enzyme Systems and Dihydrofolic Reductase*

Bertino, Joseph R.; Silber, Robert; Freeman, Monroe E.; Alenty, A.; Albrecht, Martin; Gabrio, Beverly Wescott; Huennekens, F.M.

doi:10.1172/jci104875

Cited by 91 publications

(40 citation statements)

References 27 publications

(25 reference statements)

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“…Increased levels of DNA polymerase (23), dihydrofolate reductase (24), aspartate transcarbamylase (25), dihydro-orotase (25), and dihydro-orotic dehydrogenase (25) have been observed in AML cells as compared to normal granulocytes. Rabinowitz (23) related the increased activity of DNA polymerase to immaturity of the leukemic cells by showing a decrease in polymerase activity during maturation of normal granulocytes; a similar correlation was not attempted in the other enzyme studies.…”

Section: Discussionmentioning

confidence: 99%

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

Cn³

et al. 1974

J. Clin. Invest.

111

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A B S T R A C T Cytidine deaminiase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), lhas been partially purified from normal and leukemic humiian granulocytes. The purification procedure included lheat precipitation at 700 C, ammoniunm sulfate precipitation, calcium phosphate gel ionl exchange, and Sephadex G-150 gel filtration. The enzymlle has mol xvt 51,000, isoelectric pH of 4.8, and maximlum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol.Cytidine deaminase from normal human graniulocytes lhas a greater affinity for its physiologic substrate cytidine (Km = 1.1 X 10-5 AM) than for ara-C (8.8 X 10-°MI) or 5-azaC (4.3 X 10' M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2'-deoxycytidine also exhiibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine anid deoxycytidine. No activity was observed witlh nucleotides or (leoxyniucleotides. The relative maximumlii velocity of the enzyme for cytidine and its nucleoside analogues remaiie(l constanit during purification, inidicating that a single enzynme was responsible for deaminiatioln of these substrates.Tetrahydrouridine (THU) was founid to be a strong competitive inhibitor of partially purified deamiiinase with a KT of 5.4 X 10' M.The X 10'/nmg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 X 10' U/mg proteini) or acute myelocytic leukemia (AML) cells (0.19+0.17 X 103 U/ mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of nornmal granulocvtes were studied in niormal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation ratlher than a specific enzymatic defect in leukemic cells.

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Section: Discussionmentioning

confidence: 99%

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

Cn³

et al. 1974

J. Clin. Invest.

111

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“…Plastic laboratory ware 'Performed by the Boston Medical Laboratory, Waltham, Mass. or siliconized glassware were used throughout. The leukocytes were isolated from the blood by the method of Bertino et al (6). Briefly, the blood was sedimented in a dextransaline solution, and the supernatant layer containing the leukocytes was centrifuged.…”

Section: Methodsmentioning

confidence: 99%

Alterations in thyroid hormone economy during acute infection with Diplococcus pneumoniae in the rhesus monkey

Woeber¹,

Harrison²

1971

J. Clin. Invest.

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“…The blood was centrifuged in Wintrobe hematocrit tubes at 1800 g for 30 min at room temperature., The following fractions were removed with a long 18-gauge needle: the plasma; the buffy coat, which consisted of leukocytes, platelets, and a few erythrocytes; the top one-fourth of the packed red cell column (reticulocyte-rich fraction) and the bottom one-fourth of the packed red cell column (reticulocyte-poor fraction). Contaminating erythrocytes were removed from the buffy coat fraction by osmotic lysis, as previously described (11), and the intact leukocytes and platelets were then washed, collected by centrifugation, and resuspended in a small volume of 0.15 M NaCl. Extracts of 2 These experiments were performed with the technical assistance of R. D. Carmichael, Department of Biology, Graduate School of Arts and Science, New York University.…”

Section: Methodsmentioning

confidence: 99%

Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse

Rothman¹,

Zanjani²,

Gordon³

et al. 1970

J. Clin. Invest.

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A B S T R A C T The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10-to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis.

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Studies on Normal and Leukemic Leukocytes. Iv. Tetrahydrofolate-Dependent Enzyme Systems and Dihydrofolic Reductase*

Cited by 91 publications

References 27 publications

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

Alterations in thyroid hormone economy during acute infection with Diplococcus pneumoniae in the rhesus monkey

Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse

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