Date of Award:

1998

Document Type:

Thesis

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Advisor/Chair:

Dr. Steven D. Aust

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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