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 homogenei ty. The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological as says at different protein and salt conditions. First, sedimentation eq uilibrium was preferred over the other analytical ultracentifuge metho ds as it is based on firm theoretical grounds and does not require ass umptions about the shape of the molecule. The tendency of topoisomeras e II to self-associate into dimers was confirmed and shown to depend o n both the enzyme concentration and the concentration of salt used. An alysis at five initial protein concentrations (from 0.08 to 1.05 mg/mL , i.e., 0.5-65 mu M) provided evidence for a single monomer-dimer equi librium characterized at 150 mM KCl and 20 degrees C by an association constant, K-a, of similar to 4.8 10(5) M(-1) and a Delta G degrees of similar to-7.5 kcal mol(-1). Under these conditions, for a topoisomer ase II concentration of 0.08 mg/mL (i.e., 0.5 mu M) in the ultracentri fuge cell, almost 80% of the enzyme were found dissociated. Increase o f KCl (from 80 to 400 mM) in the medium provoked a continuous change o f the association equilibrium so that a value of K-a similar to 10(5) M(-1) corresponding to Delta G degrees similar to-7 kcal mol(-1) was f ound for topoisomerase II in 400 mM KCl at 20 degrees C. Second, circu lar dichroism (CD) showed the sensitivity of the topoisomerase II seco ndary structure to salt concentration, the observed variations being a pparently dependent upon the ionic strength. For topoisomerase II in 1 50 mM KCl, i.e., optimal salt conditions for the enzyme activity, the deconvolution of CD spectra revealed a secondary structure content of 40% alpha-helix and 30% beta-turn that was the highest detected throug hout the salt dependence experiments. Third, for topoisomerase II at v arying KCl concentrations a remarkable correlation was observed betwee n the data from CD spectroscopy (secondary structure) and catalytic ac tivity. In contrast, although NaCl induced the same effects as KCl on the secondary structure of topoisomerase II, its influence on activity profile was found different, suggesting that K+ and Na+ exert distinc t effects in the biological assays. Finally, the possible stabilizatio n of topoisomerase II dimers induced by the interaction of the enzyme with its DNA sites is discussed.