Characterisation of the copper uptake mechanism and isolation of the ceruloplasmin receptor/copper transporter in human placental vesicles
1995
Abstract In this paper we have studied copper (Cu) uptake by microvillar vesicles isolated from human term placenta. We have characterised Cu uptake from CuHis 2 complexes and shown that ceruloplasmin (Cp) inhibits uptake. Inhibition is complex and variable; in one series of experiments, the V max , for uptake drops from 31.3 ± 1.2 nmol/min per mg vesicle protein without added Cp to 11.3 ± 1 nmol/min per mg vesicle protein at 91 μg/ml Cp. Similarly, the K 0.5 increases from 0.35 ± 0.08 μ M to 1.35 ± 0.25 μ M, while the n value (the Hill coefficient) falls from 1.9 ± 0.23 in the absence of Cp to 1.1 ± 0.13 In another series, Cp had no effect below concentrations of about 0.100 μg/ml and in a third series only increased K 0.5 . The variability in effect seems to be related to the specific activity of the ceruloplasmin, which in turn is related to the copper complexes of the protein. The effect is specific for Cp; apotransferrin and a 2 -macroglobulin have no effect. 67 Cu-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 μU/mg vesicle protein and a B max of 79 μU/mg vesicle protein. CuHis 2 , but not histidine alone, can block the uptake. The data can be reconciled by proposing that the binding site of the transporter is relatively small and recognises a Cu-dihistidine structure common to the low-molecular-weight complex and to the Type I and Type II coppers of ceruloplasmin. We have used these observations to develop an isolation method for the transporter and have identified it as a protein of M r 90000 which is closely associated with alkaline phosphatase. There are also two proteins of M r 45 000 and 40 000 which may be breakdown products of the larger complex. Antibodies to the 45 000 protein block Cu binding and uptake from CuHis 2 complexes, strongly implicating it as the copper transporter/ceruloplasmin receptor of human term placenta.
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