Purification and properties of cytidine deaminase from normal and leukemic granulocytes

B. A. Chabner, D. G. Johns, C. N. Coleman, J. C. Drake, W. H. Evans

Research output: Contribution to journalArticle

Abstract

Cytidine deaminase, an enzyme that catalyzes the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara C) and 5 azacytidine (5 azaC), was partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70° C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G 150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum 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 granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10 -5 M) than for ara C (8.8 x 10 -5 M) or 5 azaC (4.3 x 10 -4 M). Halogenated analogues such as 5 fluorocytidine and 5 bromo 2' deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10 -8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, mol wt, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52 ± 1.86 x 10 3 V/mg protein) than chronic myelocytic leukemia (CML) cells (1.40 ± 0.70 x 10 3 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19 ± 0.17 x 10 3 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 normal granulocytes were studied in normal 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 rather than a specific enzymatic defect in leukemic cells.

Original languageEnglish (US)
Pages (from-to)922-931
Number of pages10
JournalJournal of Clinical Investigation
Volume53
Issue number3
StatePublished - 1974
Externally publishedYes

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Cytidine Deaminase
Granulocytes
Cytidine
Enzymes
Cytarabine
Azacitidine
Deamination
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Nucleosides
Bromodeoxycytidine
Tetrahydrouridine
Bone Marrow
Ficoll
Sulfhydryl Reagents
Deoxycytidine
Proteins
Dithiothreitol
Ion Exchange
Ammonium Sulfate
Myeloid Cells

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Chabner, B. A., Johns, D. G., Coleman, C. N., Drake, J. C., & Evans, W. H. (1974). Purification and properties of cytidine deaminase from normal and leukemic granulocytes. Journal of Clinical Investigation, 53(3), 922-931.

Purification and properties of cytidine deaminase from normal and leukemic granulocytes. / Chabner, B. A.; Johns, D. G.; Coleman, C. N.; Drake, J. C.; Evans, W. H.

In: Journal of Clinical Investigation, Vol. 53, No. 3, 1974, p. 922-931.

Research output: Contribution to journalArticle

Chabner, BA, Johns, DG, Coleman, CN, Drake, JC & Evans, WH 1974, 'Purification and properties of cytidine deaminase from normal and leukemic granulocytes', Journal of Clinical Investigation, vol. 53, no. 3, pp. 922-931.
Chabner, B. A. ; Johns, D. G. ; Coleman, C. N. ; Drake, J. C. ; Evans, W. H. / Purification and properties of cytidine deaminase from normal and leukemic granulocytes. In: Journal of Clinical Investigation. 1974 ; Vol. 53, No. 3. pp. 922-931.
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abstract = "Cytidine deaminase, an enzyme that catalyzes the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara C) and 5 azacytidine (5 azaC), was partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70° C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G 150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum 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 granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10 -5 M) than for ara C (8.8 x 10 -5 M) or 5 azaC (4.3 x 10 -4 M). Halogenated analogues such as 5 fluorocytidine and 5 bromo 2' deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10 -8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, mol wt, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52 ± 1.86 x 10 3 V/mg protein) than chronic myelocytic leukemia (CML) cells (1.40 ± 0.70 x 10 3 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19 ± 0.17 x 10 3 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 normal granulocytes were studied in normal 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 rather than a specific enzymatic defect in leukemic cells.",
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N2 - Cytidine deaminase, an enzyme that catalyzes the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara C) and 5 azacytidine (5 azaC), was partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70° C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G 150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum 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 granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10 -5 M) than for ara C (8.8 x 10 -5 M) or 5 azaC (4.3 x 10 -4 M). Halogenated analogues such as 5 fluorocytidine and 5 bromo 2' deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10 -8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, mol wt, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52 ± 1.86 x 10 3 V/mg protein) than chronic myelocytic leukemia (CML) cells (1.40 ± 0.70 x 10 3 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19 ± 0.17 x 10 3 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 normal granulocytes were studied in normal 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 rather than a specific enzymatic defect in leukemic cells.

AB - Cytidine deaminase, an enzyme that catalyzes the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara C) and 5 azacytidine (5 azaC), was partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70° C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G 150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum 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 granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10 -5 M) than for ara C (8.8 x 10 -5 M) or 5 azaC (4.3 x 10 -4 M). Halogenated analogues such as 5 fluorocytidine and 5 bromo 2' deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10 -8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, mol wt, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52 ± 1.86 x 10 3 V/mg protein) than chronic myelocytic leukemia (CML) cells (1.40 ± 0.70 x 10 3 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19 ± 0.17 x 10 3 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 normal granulocytes were studied in normal 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 rather than a specific enzymatic defect in leukemic cells.

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