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Demonstration of Two Components and Association of Adenosine Diphosphate-Cytidine Diphosphate Reductase from Cultured Human Lymphoblast Cells (Molt-4F)¹

Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263

Ribonucleotide reductase was isolated from a human lymphoblast line (Molt-4F). Most of the reductase activity was present in the cytosol fraction. Two components (A and B) were found which were readily separable by deoxyguanosine triphosphate Sepharose column chromatography. Only Component B was retained on this column and could be eluted by high concentrations of KCI. Components A and B were purified further by blue Sepharose, diethylaminoethyl cellulose and phenyl-Sepharose column chromatography, as well as by sucrose gradient sedimentation. The apparent molecular weights estimated by sucrose gradient sedimentation were 100,000 for both Components A and B, and 210,000 for the nondissociated ribonucleotide reductase. The cytidine diphosphate (CDP) and adenosine diphosphate (ADP) reductase activities cochromatographed throughout the purification procedure with a constant ratio of 1.73 ± 0.19 (S.D.) Variation of the ratio of purified Component A to B led to subsequent variation in overall activity. However, the ratio of CDP to ADP enzyme activity remained constant. The enzyme activities of reconstituted purified A and B components were further characterized with reference to cation requirements. Of those divalent cations tested, magnesium ion was found to be essential for maximal enzyme activity, while calcium ion gave only partial activation. Addition of zinc or manganese ion, at concentrations higher than 0.4 mM, to the reaction mixture containing 6 mM MgCl₂ caused a marked inhibition of the enzyme activity for both ADP and CDP reduction. Spermidine and spermine can partially replace the MgCl₂ requirement for CDP and ADP reduction. The optimal concentrations of MgCl₂ and dithiothreitol were 6 and 3 mM, respectively.

¹ This work was supported by USPHS Project Grant CA-18499 and Core Grant CA-13038 from the National Cancer Institute.

² An American Leukemia Society Scholar. To whom requests for reprints should be addressed.

Received 6/ 5/78. Accepted 11/ 3/78.