PHOSPHORYLATION SITES FOR TYPE-N-II PROTEIN-KINASE IN DNA TOPOISOMERASE-I FROM CALF THYMUS

1. 1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2. 2. In vitro labeling experiments, which employed the purified enzyme [γ-32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. 5. Tryptic peptides mapping, by HV electrophoresis, indentified five major [32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 6. 6. Separation of each spot, by reverse phase HPLC, resulted in their elution at fractions 1, 2, 3, 4 and 5 with 9, 11, 16, 27 and 28% acetonitrile, respectively. 7. 7. Isolated phosphopeptides will be subjected to sequencing, to DNA-binding and transcription regulation tests; then, it will be speculated whether type N II protein kinase may contribute to the physiological regulation of DNA-topoisomerase I activity from calf thymus, as well. © 1990.