Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation.
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.
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*
D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; D000248:Adenosine Kinase; C020005:deoxyadenosine kinase; D008274:Magnesium