Purification and some properties of a deoxyribonucleoside kinase from L1210 cells.

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 (Rf 0.66), catalyzed the phosphorylation of 2'-deoxyadenosine, 2'-deoxyguanosine, 2'deoxycytidine, 9-/S-D-arabinofuranosyladenine, 9-/S-D-arabinofuranosyl-2-fluoroadenine, 1-/ß-o-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 HIM MgCI2 produced 90 to 100% inhibition of the enzyme activity. The kinase had broad speci ficity for phosphate donors; adenosine triphosphate, guanosine triphosphate, uridine triphosphate, inosine triphosphate, deoxyuridine triphosphate, and deoxythymidine triphosphate ac tively donated phosphate to deoxyadenosine as did several nucleoside triphosphate analogs. The Michaelis constant for deoxyadenosine varied with the phosphate donor; the apparent Kmvalues with adenosine triphosphate and uridine triphosphate as donor were 1.25 and 0.13 mM, respectively. Apparent Km values (0.25 mw) with deoxyuridine triphosphate and deoxy thymidine triphosphate also were lower than those obtained with adenosine triphosphate as donor, but guanosine triphos phate and inosine triphosphate gave the same constant as adenosine triphosphate. The apparent Vmaxfor phosphorylation of deoxyadenosine was severalfold higher with adenosine tri phosphate than with uridine triphosphate. The apparent Mi chaelis constants for deoxyguanosine, deoxycytidine, and cy tidine with adenosine triphosphate as phosphate donor were 1.37, 8.0, and 33 mw, respectively. Since deoxyadenosine and deoxyguanosine were the best substrates, this enzyme may be regarded as a purine deoxyribonucleoside kinase.