Partial Purification of Topoisomerase I from Mycobacterium phlei

Authors: ASLI PINAR, N. LEYLA AÇAN, NAZMİ ÖZER

Abstract: Aim: DNA topoisomerases control several cellular activities by catalyzing the relaxation of superhelices, which are produced during the replication of DNA. Investigation of the inhibitory action of the candidate substances on topoisomerase activity is widely used in drug development. The purpose of this study was to purify topoisomerase I from Mycobacterium phlei, which is closely related to Mycobacterium tuberculosis, and investigate some of its properties. Materials and Methods: Topoisomerase I from Mycobacterium phlei was partially purified with the method including homogenization followed by DNase treatment, Sephadex G50 and Heparin Sepharose column chromatography steps. Results: The enzyme was purified 31.8-fold with a yield of 16.7%. Molecular weight of the enzyme was estimated to be 125 kDa. Enzyme activity was found to be stable in the pH range of 6.0 - 8.5. ATP and spermidine were not required for the activity of the enzyme. Camptothecin, which is an inhibitor of eukaryotic topoisomerase I, did not inhibit the enzyme at the concentrations studied. Conclusions: The investigated properties of partially purified topoisomerase I from nonpathogenic strain Mycobacterium phlei were found closely related to those of Mycobacterium tuberculosis. Characterization of the enzyme that is completely purified with additional purification steps will hopefully lead to the development of selective antimycobacterial drugs.

Keywords: Mycobacterium phlei, DNA topoisomerase type I, purification, molecular properties

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