Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have reported previously that rat liver mitochondria contain a topoisomerase and have shown it to be distinct from the nuclear enzyme by its sensitivity to Berenil and ethidium bromide. We report here some additional characterization. The enzyme differs further from its nuclear counterpart in its failure to bind to ssDNA cellulose and its chromatographic behavior on Sephadex; the latter procedure yields an Mr of 44 000 for the mitochondrial and 70 000 for the nuclear enzyme. The topoisomerase is strongly associated with mitochondrial membranes; only 10% of the activity could be extracted. The pH optimum of the enzyme falls between 6.0 and 8.5, with an NaCl optimum of 0.13 M in 0.1 M Tris (pH 8.3). Dithiothreitol is required, while N-ethylmaleimide is inhibitory. Tosylphenylalanine chloromethyl ketone, a serine proteinase inhibitor, abolishes activity; another, phenylmethanesulfonyl fluoride, has no effect. Berenil, a non-intercalating drug, and four of its analogues all inhibit with up to 100-fold differences in potency. No dependence on ATP, Mg2+, or both together could be shown. Neither novobiocin nor oxolinic acid shows any inhibitory effect. Nicked circles are generated in the presence of DMSO. These three observations are consistent with the topoisomerase being of the Type I class. Positively supercoiled pBR322 DNA, whose 6-8 positive turns were generated by altering solution conditions, is relaxed by the enzyme, indicating a lack of requirement for a negatively supercoiled substrate. We have also examined a partially purified preparation of the corresponding mitochondrial enzyme from mouse L cells. This enzyme is largely similar in properties to the rat liver enzyme. In isolated mitochondria, Berenil causes biphasic alterations in [3H]dATP incorporation into DNA, 10(-4) mM stimulating 2-fold, while higher concentrations inhibit. [3H]UTP incorporation into mitochondrial RNA also follows this pattern.
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PMID:Studies on mitochondrial type I topoisomerase and on its function. 298 52

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase IIalpha levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase IIalpha poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase IIalpha monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded to identify possible sites of cisplatin adduction. High-pressure liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry showed that topoisomerase IIalpha contained at least five free cysteines (170, 216, 300, 392, and 405) and two disulfide-bonded cysteine pairs (427-455 and 997-1008). Cysteine 733 was also disulfide-bonded, but its partner cysteine could not be identified. Cisplatin antagonized the formation of a fluorescence adduct between topoisomerase IIalpha and the sulfhydryl-reactive maleimide reagent 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic acid methyl ester (ThioGlo-1). Dithiothreitol, which was shown by spectrophotometry to react rapidly with cisplatin (6-min half-time), diminished the capacity of cisplatin to interfere with ThioGlo-1 binding to topoisomerase IIalpha. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase IIalpha through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase IIalpha.
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PMID:Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha. 1560 6