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Purification and Some Properties of a Deoxyribonucleoside Kinase from L1210 Cells¹

Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255

A nucleoside kinase has been purified about 1680-fold from L1210 leukemia cells with deoxyadenosine as the phosphate acceptor and with adenosine triphosphate as phosphate donor. The molecular weight of the enzyme, determined by Sephadex G-200 column chromatography, was found to be about 100,000. Purified enzyme, after the final step of purification and also after disc gel electrophoresis (R_f 0.66), catalyzed the phosphorylation of 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 9-ß-D-arabinofuranosyladenine, 9-ß-D-arabinofuranosyl-2-fluoroadenine, 1-ß-D-arabinofuranosylcytosine, and cytidine.

The enzyme exhibited a broad pH optimum ranging from 7.8 to 8.7. Magnesium ion and nucleoside triphosphates were essential for activity. The addition of Ni²⁺, Zn²⁺, or Co²⁺ to the reaction mixture containing 2 mM MgCl₂ produced 90 to 100% inhibition of the enzyme activity. The kinase had broad specificity for phosphate donors; adenosine triphosphate, guanosine triphosphate, uridine triphosphate, inosine triphosphate, deoxyuridine triphosphate, and deoxythymidine triphosphate actively donated phosphate to deoxyadenosine as did several nucleoside triphosphate analogs. The Michaelis constant for deoxyadenosine varied with the phosphate donor; the apparent K_m values with adenosine triphosphate and uridine triphosphate as donor were 1.25 and 0.13 mM, respectively. Apparent K_m values (0.25 mM) with deoxyuridine triphosphate and deoxythymidine triphosphate also were lower than those obtained with adenosine triphosphate as donor, but guanosine triphosphate and inosine triphosphate gave the same constant as adenosine triphosphate. The apparent V_max for phosphorylation of deoxyadenosine was severalfold higher with adenosine triphosphate than with uridine triphosphate. The apparent Michaelis constants for deoxyguanosine, deoxycytidine, and cytidine with adenosine triphosphate as phosphate donor were 1.37, 8.0, and 33 mM, respectively. Since deoxyadenosine and deoxyguanosine were the best substrates, this enzyme may be regarded as a purine deoxyribonucleoside kinase.

¹ This investigation was supported by USPHS Grant RO1-CA-23155 awarded by the National Cancer Institute and Grant SO7-RR-05676, Division of Research Resources, Department of Health and Human Services.

² To whom requests for reprints should be addressed.

Received 12/ 3/81. Accepted 5/ 6/82.