Thymidylate kinase from Acetabularia. I. Properties of the enzyme

The occurrence of a dTMP kinase as well as a dTDP kinase in Acetabularia mediterranea has been demonstrated. A test system was developed by which it was possible to estimate the enzyme activity in an individual Acetabularia cell or even in a cell fragment. The enzyme catalyses the phosphorylation of dTMP in the presence of ATP. In the test system described, dTTP is formed as well as dTDP. This indicates that there is also a dTDP kinase present in the enzyme preparation. Characteristics of the enzyme such as pH optima at pH 7.0 and 8.75, a temperature optimum at 45 degrees C, a Km value of 3.3 X 10(-6) M and a high specificity for ATP were established. In homogenates that were preserved at -70 degrees C the enzyme activity was retained even after many weeks. Freezing at -70 degrees C and then thawing resulted in an increase in enzyme activity. The enzyme was inhibited by low concentrations of dTTP. After centrifugation of homogenates the greater part of the enzyme activity was found in the sediment. From the observation that purified chloroplast preparations contained most of the dTMP kinase activity, and that chlorophyll and enzyme activity cosedimented in a linear sucrose gradient, it was concluded that the enzyme is located in the chloroplasts.