title: Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation.
abstract: The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 microM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 microM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 microM deoxyguanosine was inhibited approximately 90% by 10 microM deoxycytidine or dCTP and was inhibited 70% by 200 microM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 microM, phosphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 microM, was partially inhibited by 200 microM adenosine, 34%; 200 microM deoxyguanosine, 10%; and 100 microM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 microM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.
mesh_terms: D000248:Adenosine Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002499:Centrifugation, Density Gradient; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
chemical_list: D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; D000248:Adenosine Kinase; C020005:deoxyadenosine kinase; D008274:Magnesium