LOADING OF IRON INTO RECOMBINANT RAT-LIVER FERRITIN HETEROPOLYMERS BYCERULOPLASMIN

We have reported previously that the heavy chain of ferritin is requir ed for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S . D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for cerulop lasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferrit in were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infectious Autograp ha californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the in fection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light ( L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H ch ain in the two heteropolymers. The extent of iron incorporation decrea sed with additional ceruloplasmin. Iron incorporation into rat liver f erritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up t o 8:1. Iron loading into horse spleen ferritin, found to have one H ch ain, appeared similar to that for recombinant ferritin, having only on e H chain. Therefore, we propose that the optimal molar ratio of cerul oplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin . These results also suggest that the iron loading channel is containe d within a single H chain subunit. (C) 1997 Academic Press.