Properties of an affinity-column-purified human deoxycytidylate deaminase.
Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.
Maley
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Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Lobo
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AP
Maley
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F
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003844:DCMP Deaminase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D003843:Deoxycytidine Monophosphate / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology
D000906:Antibodies; D013942:Thymine Nucleotides; D003843:Deoxycytidine Monophosphate; D003844:DCMP Deaminase; C024157:thymidine 5'-triphosphate