Hilton M, Spenser D C, Ross P, Ramsey A, McArdle H J
Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Scotland, UK.
Biochim Biophys Acta. 1995 Oct 19;1245(2):153-60. doi: 10.1016/0304-4165(95)00084-o.
In this paper we have studied copper (Cu) uptake by microvillar vesicles isolated from human term placenta. We have characterised Cu uptake from CuHis2 complexes and shown that ceruloplasmin (Cp) inhibits uptake. Inhibition is complex and variable; in one series of experiments, the Vmax 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 micrograms/ml Cp. Similarly, the K0.5 increases from 0.35 +/- 0.08 microM to 1.35 +/- 0.25 microM, 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 100 micrograms/ml and in a third series only increased K0.5. 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 a2-macroglobulin have no effect. 67Cu-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 microU/mg vesicle protein and a Bmax of 79 microU/mg vesicle protein. CuHis2, 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) 90,000 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 CuHis2 complexes, strongly implicating it as the copper transporter/ceruloplasmin receptor of human term placenta.
在本文中，我们研究了从足月人胎盘分离的微绒毛小泡对铜（Cu）的摄取。我们对从CuHis2复合物摄取铜进行了表征，并表明铜蓝蛋白（Cp）抑制摄取。抑制作用复杂且多变；在一系列实验中，摄取的Vmax从每毫克小泡蛋白31.3±1.2 nmol/分钟（无添加Cp时）降至每毫克小泡蛋白11.3±1 nmol/分钟（在91微克/毫升Cp时）。同样，K0.5从0.35±0.08微摩尔增加到1.35±0.25微摩尔，而n值（希尔系数）从无Cp时的1.9±0.23降至1.1±0.13。在另一系列中，Cp在浓度约100微克/毫升以下无作用，在第三系列中仅增加了K0.5。作用的变异性似乎与铜蓝蛋白的比活性有关，而铜蓝蛋白的比活性又与该蛋白的铜复合物有关。这种作用对Cp具有特异性；脱铁转铁蛋白和α2-巨球蛋白无作用。67Cu标记的铜蓝蛋白以2.8微单位/毫克小泡蛋白的亲和力和79微单位/毫克小泡蛋白的Bmax特异性结合足月胎盘的小泡。CuHis2而非单独的组氨酸可阻断摄取。通过提出转运体的结合位点相对较小且识别低分子量复合物以及铜蓝蛋白的I型和II型铜共有的Cu-二组氨酸结构，可以使这些数据得到合理的解释。我们利用这些观察结果开发了一种转运体的分离方法，并将其鉴定为一种分子量为90,000的蛋白质，它与碱性磷酸酶密切相关。还有两种分子量为45,000和40,000的蛋白质，它们可能是较大复合物的降解产物。针对45,000蛋白的抗体可阻断CuHis2复合物的铜结合和摄取，强烈表明它是人足月胎盘的铜转运体/铜蓝蛋白受体。
