PURIFICATION AND PROPERIES OF HUMAN DEOXYCYTIDINE KINASE: 17

Abstract

Deoxycytidine kinase is the principal deoxynucleoside salvage enzyme in humans. It plays a central role in regulation of DNA precursor metabolism in lymphocytes. The enzyme is involved in the pathogenicity of certain inherited immunodeficiency diseases, associated with high deoxynucleoside levels. We have purified the enzyme from human T-leukemic cells in culture (CEM-cells) and from human leukemic spleen (chronical lymphatic leukemia of B-cell type). A similar specific activity was found in crude extracts from both CEM-cells and leukemic spleen. Therefore the latter constitutes a good source for purification of the enzyme. From 1000 mg spleen approximately 10 μg purified enzyme was obtained, and thus the cell content of the enzyme is very low. The purification involves ammonium sulfate precipitation, DEAE-Sephadex chromatography and affinity chromatography on dTTP-Sepharose. The preparation is now highly purified and on SDS-gel electrophoresis 3-4 bands are apparent. In the purified fractions, deoxyguanosine also served as a substrate for the enzyme but with a 50-fold higher K_m value than deoxycytidine.

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