Evidence for a protein-protein complex during iron loading into ferritin by ceruloplasmin.
The formation of a protein-protein complex for the loading of iron into ferritin by ceruloplasmin was investigated. Ferritin stimulated the ferroxidase activity of ceruloplasmin unless the ferritin was fully loaded, in which case it inhibited the ferroxidase activity of ceruloplasmin. The apparent association constant for the interaction of ferritin and ceruloplasmin was 24 nM. Isothermal titration calorimetry indicated that the interaction of ceruloplasmin and ferritin was endothermic, driven by positive changes in entropy. The association constants for complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7 x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized forms of ceruloplasmin, respectively. The oxidized form of ceruloplasmin was retained on an affinity column with ferritin immobilized as the ligand and remained bound to the column with mobile phases of increased hydrophobicity, but was eluted with increased ionic strength. The ability of ceruloplasmin to remain bound to the affinity resin was affected by the species from which ceruloplasmin was isolated. Gradient ultracentrifugation also provided evidence that the two proteins were associated, since ferritin promoted migration of ceruloplasmin through the gradient. Including ferrous iron in the gradient resulted in reduction of ceruloplasmin and increased the mobility of ceruloplasmin with ferritin. These data provide evidence that ferritin and ceruloplasmin form a protein-protein complex during iron loading into ferritin, which may limit redox cycling of iron in vivo.
Reilly
C A
CA
Biotechnology Center, Utah State University, Logan 84322-4705, USA.
Sorlie
M
M
Aust
S D
SD
D000818:Animals; D001052:Apoferritins / Q000378:metabolism; D002151:Calorimetry; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002570:Ceruloplasmin / Q000378:metabolism*; D002846:Chromatography, Affinity; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D005293:Ferritins / Q000378:metabolism* / Q000494:pharmacology; D005990:Glycerol; D006736:Horses; D007501:Iron / Q000378:metabolism*; D046911:Macromolecular Substances; D051381:Rats
D046911:Macromolecular Substances; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin; D005990:Glycerol