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Effects of Nucleoside Triphosphates on Human Ribonucleotide Reductase from Molt-4F Cells¹

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

The effects of nucleoside triphosphates on various nucleoside diphosphate reductions catalyzed by a highly purified ribonucleotide reductase from Molt-4F cultured human cells were examined. It was found that deoxyadenosine 5'-triphosphate strongly inhibited all four reductions. The reduction of pyrimidine nucleoside diphosphate in the presence of an activator [adenosine 5'-triphosphate (ATP)] was inhibited in a noncompetitive manner with respect to ATP by deoxyguanosine 5'-triphosphate (dGTP) and deoxythymidine 5'-triphosphate (dTTP). For cytidine 5'-diphosphate reduction, the value of the K_i intercept for dGTP was 47 µM and for dTTP, it was 270 µM; the K_i slope was 25 µM for dGTP, 100 µM for dTTP. Similarly, for uridine diphosphate reduction, the K_i intercept was 4.3 µM for dGTP, 25 µM for dTTP. The K_i slope was 1.5 µM for dGTP and 9 µM for dTTP. The reduction of ADP in the presence of its activator (dGTP) was inhibited noncompetitively by dTTP. The values of K_i intercept and slope of dTTP for adenosine 5'-diphosphate (ADP) reduction were 1.8 and 0.9 mM, respectively. Although guanosine 5'-triphosphate (GTP) and dGTP were found to serve equally well as activators for ADP reductions with the same apparent K_a and V_max, the inhibition pattern of GTP and dGTP on the enzyme activity for cytidine 5'-diphosphate reduction was different. ATP was found to be an accessory activator for ADP reduction due to the fact that ATP at 1.0 or 0.3 mM concentration decreased the apparent K_a of GTP for ADP reduction from 1.1 to 0.14 or 0.08 mM, respectively. In the absence of ATP, the V_max for ADP reduction was increased 2-fold. ATP at a concentration of 1.0 or 0.3 mM also changed the apparent K_a of dTTP for guanosine 5'-diphosphate reduction from 1.25 to 0.9 or 0.6 µM, respectively, but V_max for guanosine 5'-diphosphate reduction was increased.

GTP at a concentration of 0.3 or 0.5 mM decreased the apparent K_a's of ATP for pyrimidine nucleoside reduction. At these concentrations of GTP, the V_max for cytidine 5'-diphosphate reduction was decreased 7 and 12%, and the V_max for uridine 5'-diphosphate reduction was decreased 33 and 45% when they were compared with those in the absence of GTP. Therefore, a change in any of these nucleotide concentrations could lead to changes in ribonucleotide reductase activity. ATP emerges as a most important factor in controlling the reduction of all four ribonucleoside diphosphates.

¹ This work was supported by USPHS Project Grant CA-18499 and Core Grant CA-13038 from the Division of Cancer Treatment, National Cancer Institute, NIH, Department of Health, Education, and Welfare.

² Present address: Biochemistry Department, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Ala. 35205.

³ An American Leukemia Society Scholar. To whom requests for reprints should be addressed. Present address: Department of Pharmacology, University of North Carolina, Chapel Hill, N. C. 27514.

Received 6/22/79. Accepted 9/17/79.