Purification and properties of adenosine kinase from rat liver: separation from deoxyadenosine kinase activity.
Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS-polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 mumol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2'-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (approximately 1.5%) under optimal conditions (pH 7.5) vs 2'-deoxy-adenosine, the lowest yet reported for such a preparation, with a Km of 670 microM, as compared to 0.3 microM for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a Ki of 70 microM. Competitive inhibition was also exhibited by cordycepin (3'-deoxyadenosine) with a Ki of 150 microM. A more potent competitive inhibitor was tubercidin, the Ki for which was 1.9 microM.
Drabikowska
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Halec
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D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011506:Proteins / Q000032:analysis; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000248:Adenosine Kinase; C020005:deoxyadenosine kinase; D008274:Magnesium