Copper Binding of Nuclear RNA- and DNA-Binding Proteins p54^nrb/NonO and PSF

抄録

The biological function of nuclear copper in mammalian cells is poorly understood because little is known about specific binding protein(s) for nuclear copper. We recently purified that p54^nrb/Non-POU-domain-containing, octamer binding protein (p54^nrb/NonO) and polypyrimidine tract-binding protein-associated splicing factor (PSF), which are RNA- and DNA- binding multifunctinal nuclear proteins, from nuclear extracts of mice brain by immobilized metal affinity chromatography with copper ion (Cu-IMAC). In the present study, we purified both overexpressed p54^nrb/NonO and PSF in HEK 293 cells by Cu-IMAC. Scatchard plot analysis of copper binding to E. coli cells expressing recombinant p54^nrb/NonO protein revealed that the dissociation constant was 0.13 μM and the maximum binding number was 2.5 atoms per molecule of the recombinant protein. Using ultrafiltration assay, zinc ion is also shown to bind to the recombinant protein. However, zinc ion did not competitively inhibit the binding of copper to the recombinant protein. Moreover, p54^nrb/NonO protein did not bind specifically to other divalent heavy metals such as Cd²⁺, Mn²⁺, Pb²⁺, and Co²⁺.