Expression, purification, and metal binding properties of the N-terminal domain from the wilson disease putative copper-transporting ATPase (ATP7B).
The putative copper binding domain from the copper-transporting ATPase implicated in Wilson disease (ATP7B) has been expressed and purified as a fusion to glutathione S-transferase. Immobilized metal ion affinity chromatography revealed that the fusion protein is able to bind to columns charged with different transition metals with varying affinities as follows: Cu(II)>>Zn(II)>Ni(II)>Co(II). The fusion protein did not bind to columns charged with Fe(II) or Fe(III). 65Zinc(II) blotting analysis showed that the domain is able to bind Zn(II) over a range of pH values from 6.5 to 9.0. Competition 65Zn(II) blotting showed that Cd(II), Hg(II), Au(III), and Fe(III) can successfully compete with Zn(II), at comparable concentrations, for binding to the domain. In contrast, the domain had little or no affinity for Ca(II), Mg(II), Mn(II), and Ni(II) relative to copper. Neutron activation analysis of the copper bound to the domain showed a copper:protein ratio of 6.5-7.3:1. Both Cu(II) and Cu(I) were found to have a higher affinity for the domain relative to Zn(II). In addition, a sharp, reproducible transition was only observed in competition experiments with copper, which may suggest that copper binding has some degree of cooperativity.
DiDonato
M
M
Department of Biochemistry Research, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada.
Narindrasorasak
S
S
Forbes
J R
JR
Cox
D W
DW
Sarkar
B
B
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D027682:Cation Transport Proteins; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D004926:Escherichia coli; D006527:Hepatolenticular Degeneration / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D019216:Metals, Heavy / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D019216:Metals, Heavy; D003035:Cobalt; D003300:Copper; D009532:Nickel; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D015032:Zinc