Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca).
The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA.
Badaracco
G
G
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Italy.
Landsberger
N
N
Benfante
R
R
D000818:Animals; D000906:Antibodies; D001156:Artemia / Q000201:enzymology*; D003086:Bacteriocin Plasmids; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D000906:Antibodies; D007106:Immune Sera; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004264:DNA Topoisomerases, Type I