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in extra-cellular iron between cell lines after normalizing with 3H-thymidine (Figure
9A). Estimation of cell-associated
59
Fe after 16 hr of chase shows more
59
Fe in PrP
C
and PrP
102L
, and significantly less in PrP
Δ51-89
and PrP
Δ23-89
compared to M17 lysates as
observed in Figure 1 above (Figure 9B). However, the fold difference in ferritin iron
content between M17 and other cell lines is significantly less after 16 hr of chase, and
represents steady state levels of iron content in each cell line. Evaluation of possible
ferroxidase activity of recombinant PrP using plasma as a positive control yielded
negative results (Figure 9C). Though informative, this result does not rule out possible
ferroxidase activity of cell-associated PrP, a technically challenging assay that has
yielded inconclusive results (data not shown).
Figure 9.
PrP is not involved in the export of iron from cells. (A) Cells expressing PrP
C
, PrP
Δ
51-89
,
PrP
Δ
23-89
, and PrP
102L
were radiolabeled with
59
FeCl
3
, washed with PBS supplemented with DFO, and
chased in complete medium for 30, 60, 90, 120 min, and 16 hr. At the indicated time points equal
aliquots of medium samples were quantified in a
-counter. Estimation of released
59
Fe does not
show a significant difference between the indicated cell lines at any time point. n = 6 experiments in
triplicate. (B) Cell associated
59
Fe after 16 hr of chase reflects the ferritin iron content of each cell line
noted in Figure 1 above, though the difference between cell lines is significantly less. (C) Possible
ferroxidase activity of recombinant PrP was measured using the established colorimetric method [44]
with modifications. Negative controls included water and albumin supplemented with copper, and
positive controls included plasma in the absence or presence of copper. Recombinant PrP does not
show detectable ferroxidase activity either in the absence or presence of copper, whereas plasma
shows a robust reaction under similar conditions.
γ
