The identification of the ceruloplasmin region interacting with the copper transferring menkes ATPase

The interaction was studied of ceruloplasmin (Cp, EC 1.16.3.1), a copper-containing plasma protein, with two synthetic peptides P15 and P16 whose structures correlate with those of the noncytosolic regions of the copper transfer P1 type ATPase (ATP7A), apparently encoded by the Menkes disease gene (Atp7a). Pentadecapeptide P15 and hexadecapeptide P16 were synthesized using the solid phase method. They correspond to fragments of two extracellular loops ATP7A, of which one loop is apparently involved in the copper ion transfer (P16) whereas the otheris not (P15). The protein footprinting showed that P16 binds to a fragment of the ceruloplasmin domain 6. Kinetics of the ceruloplasmin-P16 binding was studied by affinity chromatography on P16 immobilized on a macroporous disk, and theKd value (1.5¢10−6 M) of this interaction was determined. The ATP7A involvement in the copper ion transfer to nonhepatocyte cells is discussed.