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Metallothionein is a small, highly conserved
protein that has been purported to play a central role in the cellular
homeostasis and detoxification of metals. The protein has a highly
unusual amino acid composition which is comprised of about 30% cysteine.
These cysteines serve to ligate between 7-13 metal atoms depending upon
the type of metal being bound.
Recent evidence has indicated that the binding
affinity of the thiol clusters for metals in metallothionein are strongly
affected by redox conditions. Under oxidizing conditions, the thiols
form disulfide bridges releasing the metal. Conversely, under reducing
conditions the structure of the thiol clusters are maintained and the protein
shows a high affinity for the metal. Theoretically therefore, metallothionein
could act as a detoxifying protein for excess metals under reducing conditions
and a source for physiologically important metals under oxidizing conditions.
Metals released via metallothionein in this manner could then be donated
to apo-metalloproteins for their activation. Based upon earlier work
by others, it is now thought that the donation of metals from metallothionein
may be indirect and facilitated by an intermediary molecule such as GSH.
One of the attractive features of this proposed model is that GSH in itself
is a redox sensitive molecule being transformed to GSSG under oxidizing
conditions. The redox potential of GSSG is sufficient to oxidize
metallothionein and the metals released by the metallothionein can be directly
and quickly scavenged by the GSH formed via the redox couple. By
facilitating both oxidation and metal sequestration, GSH would limit the
potential for non-specific binding of the metal to inappropriate target
molecules. Presumably the GSH-metal complex is the moiety responsible
for the activation of the various apo-metalloproteins.
Another feature of this model is that the
reverse reaction could be mediated under reducing conditions. This
reaction is theoretically made possible because of the high affinity shown
by metallothionein for metals under reducing conditions (eg. Cu Kd = 1018).
To empirically test this proposition it is necessary to first generate
apo-metallothionein and then subsequently incubate the preparation with
native metalloproteins in the presence of GSH under reducing conditions.
This poster specifically describes the generation of apo-metallothionein.
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