Phosphorylation sites for type N II protein kinase in DNA-topoisomerase I from calf thymus.
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. In vitro labeling experiments, which employed the purified enzyme [gamma-32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. Tryptic peptides mapping, by HV electrophoresis, identified five major [32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 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. 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.
Coderoni
S
S
Department of Cell Biology, University of Camerino, Italy.
Paparelli
M
M
Gianfranceschi
G L
GL
D000818:Animals; D002417:Cattle; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism*; D013950:Thymus Gland / Q000032:analysis / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D010761:Phosphorus Radioisotopes; D011494:Protein Kinases; C061570:protein kinase N; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D014357:Trypsin; D004264:DNA Topoisomerases, Type I