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Temporal changes in the isotopic signatures of 63Cu and 65Cu have been used to estimate the rates of turnover and accumulation of
copper in Cu-containing proteins isolated from the visceral complex of specimens of the marine prosobranch gastropod
mollusc, Littorina littorea, subjected to elevated levels of Cu in the external medium. Groups of animals were exposed for 8 weeks to seawater
containing stable isotopic 65Cu and then transferred to media containing stable isotopic 63Cu. The free ion activity of each isotope was
maintained at ecologically relevant levels of 10-11 M using a NTA based metal buffer system.
Tandem linked size exclusion-ion
exchange HPLC coupled to ICP-MS was used to fractionate and subsequently quantify the isotopic abundance of 63Cu and 65Cu and
the content of Zn, Mo, Mn, Cd, Co, Ag, Hg and Pb in cytosolic proteins.
Cytosolic Cu could be fractionated by size exclusion into
two ligand pools with apparent molecular weights of >300 kDa and approximately 17 kDa. The latter pool was inducible by exposure
to Cu and showed a higher rate of Cu accumulation/turnover than the high molecular weight moieties. Isotopic calculations indicate that
the Cu associated with this induced pool was derived from both intrinsic and extrinsic sources.
Another unusual feature of this low
molecular weight protein pool is its apparent ability to bind a number of metals other than Cu including Zn, Cd, Pb, Mn and Co. Further
fractionation in a second dimension using ion exchange HPLC revealed that the pool comprised of a heterogeneous mixture of proteins.
Cu was associated with a single major protein that also bound Cd, Zn, Co and Pb. Three additional Cd containing proteins were
observed, the major of which also bound Pb, Mn and minor quantities of Zn. The significance of these findings in metal homeostasis and
detoxification is discussed.
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