Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides.
An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride; D009711:Nucleotides / Q000494:pharmacology*; D010053:Ovary; D012264:Ribonucleotide Reductases / Q000378:metabolism*
D009711:Nucleotides; D015636:Magnesium Chloride; D012264:Ribonucleotide Reductases; D008274:Magnesium; D004229:Dithiothreitol