Uracil DNA-glycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients

Abstract

The enzyme uracil DNA-glycosylase has been purified from blast cells of patients with acute myelocytic leukemia. A 1000-fold purification has been achieved and the enzyme appears highly enriched for the uracil glycosylase activity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the purified enzyme is 30,000. Uracil DNA-glycosylase exhibits activity in the absence of any added metal and the addition of MgCl2, MnCl2, CaCl2, NaCl, or KCl causes inhibition. EDTA as well as EGTA can inhibit enzyme activity. An interesting finding is the biphasic effect of spermine. At a concentration of 25 microM, spermine will cause a 2.5-fold activation of enzyme activity, whereas at concentrations of 100 microM and higher, spermine will inhibit enzyme activity. An Arrhenius plot of glycosylase activity in the presence of 25 microM spermine shows a biphasic curve with the transition temperature being 36 degrees C. Initial velocity studies in the presence of varying concentrations of spermine indicate a change in both the apparent Km and Vmax of the enzyme. Various uracil analogs were tested to establish a structure-activity relationship for this enzyme. It appears from this data that uracil DNA-glycosylase is very specific for uracil moieties. Uracil, acting as a product inhibitor, gives a Ki value of 220 microM.