Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions.
Topoisomerase II overexpressed in yeast was purified to near homogeneity. The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological assays at different protein and salt conditions. First, sedimentation equilibrium was preferred over the other analytical ultracentifuge methods as it is based on firm theoretical grounds and does not require assumptions about the shape of the molecule. The tendency of topoisomerase II to self-associate into dimers was confirmed and shown to depend on both the enzyme concentration and the concentration of salt used. Analysis at five initial protein concentrations (from 0.08 to 1.05 mg/mL, i.e., 0.5-65 microM) provided evidence for a single monomer-dimer equilibrium characterized at 150 mM KCl and 20 degrees C by an association constant, Ka, of approximately 4.8 10(5) M-1 and a delta G degree of approximately -7.5 kcal mol-1. Under these conditions, for a topoisomerase II concentration of 0.08 mg/mL (i.e., 0.5 microM) in the ultracentrifuge cell, almost 80% of the enzyme were found dissociated. Increase of KCl (from 80 to 400 mM) in the medium provoked a continuous change of the association equilibrium so that a value of Ka approximately 10(5) M-1 corresponding to delta G degree approximately -7 kcal mol-1 was found for topoisomerase II in 400 mM KCl at 20 degrees C. Second, circular dichroism (CD) showed the sensitivity of the topoisomerase II secondary structure to salt concentration, the observed variations being apparently dependent upon the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Lamhasni
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Département de Biologie Structurale, CNRS URA 147, INSERM U 140, Institut Gustave Roussy, Villejuif, France.
Larsen
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AK
Barray
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M
Monnot
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M
Delain
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E
Fermandjian
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D002384:Catalysis; D002942:Circular Dichroism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D009994:Osmolar Concentration; D011189:Potassium Chloride; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D011189:Potassium Chloride; D004250:DNA Topoisomerases, Type II