CHARACTERIZATION OF THE COPPER UPTAKE MECHANISM AND ISOLATION OF THE CERULOPLASMIN RECEPTOR COPPER TRANSPORTER IN HUMAN PLACENTAL VESICLES

In this paper we have studied copper (Cu) uptake by microvillar vesicl es isolated from human term placenta. We have characterised Cu uptake from CuHis(2) complexes and shown that ceruloplasmin (Cp) inhibits upt ake. 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 mu g/ml Cp. Similarly, the K-0.5 increases from 0.35 +/- 0.08 m u M to 1.35 +/- 0.25 mu M, while the n value (the Hill coefficient) fa lls 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 100 mu g/ml and in a third series only increased K-0.5. The variability in effect see ms to be related to the specific activity of the ceruloplasmin, which in rum is related to the copper complexes of the protein. The effect i s specific for Cp; apotransferrin and a(2)-macroglobulin have no effec t. Cu-67-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 mu U/mg vesicle protein and a B-max of 79 mu U/mg vesicle protein. CuHis(2), but not histidine alone, can block the uptake. The data can be reconciled by proposing that max the binding site of the transporter is relatively small and recognises a Cu-dihistidine structure common to the low-molecular-weight complex an d to the Type I and Type II coppers of ceruloplasmin. We have used the se observations to develop an isolation method for the transporter and have identified it as a protein of M(r) 90 000 which is closely assoc iated with alkaline phosphatase. There are also two proteins of M(r) 4 5 000 and 40 000 which may be breakdown products of the larger complex . Antibodies to the 45 000 protein block Cu binding and uptake from Cu His(2) complexes, strongly implicating it as the copper transporter/ce ruloplasmin receptor of human term placenta.