Measurement of nucleoside kinases in crude tissue extracts.
The measurements of deoxyadenosine kinase, adenosine kinase, and deoxycytidine kinase were examined in human placental cytosol to achieve a valid and reliable assay linear with time and protein. Our studies confirm the need to inhibit deaminase enzymes, since deoxyadenosine and deoxycytidine undergo extensive deamination and phosphorolysis. The use of a uniformly labeled nucleoside substrate introduced an artifact because the chromatographic behavior of the deoxyribose-1-phosphate, formed during the assay, was difficult to distinguish from the deoxynucleoside phosphate product. Accurate product identification was also essential. Finally, the substitution of GTP in place of ATP as the phosphate donor, the addition of a sulfhydryl reducing agent and a monovalent cation need to be considered when an assay is optimized. The use of these methods have lead to valid assays in placental cytosol that are linear with time and protein. Consideration of these important principles are necessary when establishing a valid and reliable nucleoside kinase assay in a crude tissue preparation.
D000248:Adenosine Kinase / Q000032:analysis; D002414:Cations, Monovalent / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000032:analysis; D005260:Female; D006801:Humans; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000032:analysis*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013438:Sulfhydryl Compounds
D002414:Cations, Monovalent; D010710:Phosphates; D013438:Sulfhydryl Compounds; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000248:Adenosine Kinase; D003842:Deoxycytidine Kinase; C020005:deoxyadenosine kinase; D009700:Nucleoside Deaminases