Purification and characterization of a eukaryotic type 1 topoisomerase from pea chloroplast.
A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.
Mukherjee
S K
SK
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Reddy
M K
MK
Kumar
D
D
Tewari
K K
KK
D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010946:Plants, Medicinal; D013379:Substrate Specificity
D004278:DNA, Superhelical; D010761:Phosphorus Radioisotopes; D004264:DNA Topoisomerases, Type I