Date of Award:

5-1998

Document Type:

Thesis

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Committee Chair(s)

Steven D. Aust

Committee

Steven D. Aust

Committee

Jeff Broadbent

Committee

Daren P. Cornforth

Committee

Thomas A. Grover

Committee

Deloy G. Hendricks

Abstract

Ferritin is an iron storage protein consisting of H and L chains to form a 24-subunit heropolymer. Ceruloplamin oxidizes Fe(II) and then loads the iron into ferritin. This research was conducted to determine which ferritin subunit is involved and whether a proposed iron-loading channel is required for iron loading by ceruloplasmin.

Recombinant rat liver H and L chain ferritin homopolymers, designated as rH-Ft and rL-Ft, respectively, were produced using insect cell-baculovirus and Escherichia coli expression systems. The expressed rH-Ft strongly suppressed the growth of the host. The rH-Ft expressed in the E. coli contained approximately 150 iron atoms/ferritin and was observed to have protein damage, which was found to affect iron-loading by ceruloplasmin. The ferritin expressed in the E. coli system apparently was not proper for this iron loading study. Alternatively, the ferritins expressed in the insect cell-baculovirus system were utilized for this purpose. Ceruloplasmin was able to load iron into the rH-Ft, but not the rL-Ft. The initial rate of loading iron into the rH-Ft by ceruloplasmin was similar to that of native rat liver ferritin heteropolymer. Both the rH-Ft and the native rat liver ferritin could be maximally loaded with iron by ceruloplasmin up to 2,500 iron atoms/ferritin. When the rH-Ft or the native ferritin was present, the ferroxidase activity of ceruloplasmin was enhanced. No such enhancement was observed in the presence of the rL-Ft. This suggests that ceruloplasmin only associates with the ferritin H, but not L, chain during iron loading.

The role of an a-helix bundle channel in iron loading by ceruloplasmin was investigated by using sitedirected mutagenesis. The channel in the rH-Ft was closed by mutation E62K and H65G to form a K62 to El07 salt bridge, which is thought to exist in the L chain. Conversely, the salt bridge in the channel of the L chain was removed by mutation K58E and G61H to form a channel similar to that in the four-a-helix bundle of the H chain. The initial rate of loading iron into the rL-FT mutant by ceruloplasmin was 50% of that for loading iron into the rH-Ft. When 500 atoms of iron per ferritin were used for loading, 98% loaded into the rH-Ft by ceruloplasmin in 5 minutes, but only 30% loaded into the rL-Ft mutant in the same time. The ferroxidase activity of ceruloplasmin was enhanced in the presence of the rHFt and its mutant, but not in the presence of the rL-Ft or its mutant. These results indicate that the association of ceruloplasmin and ferritin is required and the a-helix bundle channel is a channel for iron loading.

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