Abstract
Albumin and amino acid bound Cu are the readily accessible forms of plasma Cu with a half-life of ∼10 min [1]. Hepatic uptake largely accounts for this rapid clearance of exchangeable plasma copper [2]. We have recently characterized the kinetics of a Cu-specific transport protein of rat hepatocytes [3] and here report the effect of plasma components.
Albumin markedly inhibits
64Cu(II) uptake at up to 10:1 molar excess of Cu. In the presence of albumin, the nonlinear Lineweaver-Burk plots obtained converge to the same V
max and K
m parameters as for free Cu which indicates inhibition by a substrate-removal mechanism. Histidine facilitates albumin-inhibited Cu(II) uptake, but rates of Cu uptake in the presence of histidine do not exceed the rates Cu(II). Several (10) amino acids were tested including Thr and Gln which have been detected in Cu-complexes isolated from plasma [4], but only histidine facilitated albumin-inhibited Cu-uptake. Moreover, the facilitating activity of a low molecular weight 5000 daltons) rat plasma fraction was accounted for by its histidine content. The tripeptide, Gly-His-Lys which was reported to facilitate Cu uptake in hepatoma cell cultures [5] had inhibitory activity similar to albumin.
Albumin was dialyzed with
64Cu [
3H]-His, and the transport activities of the albumin-containing and albumin-free fractions in equilibrium were compared. Transport activity was completely accounted for within the excess histidine plus
64Cu(II) fraction. At pH 7.4, the predominant species was His
2Cu(II) [6]. This complex exhibited the identical V
max, but higher (20
vs. 10μM) K
m as free Cu(II). Given the stability constant of His
2Cu(II) (β
102- 10
18) [6], the transport activity of the complex cannot be accounted for by free Cu(II) in equilibrium with the complex. Copper uptake experiments with [
3H]-His
2
64Cu(II) showed that Cu and His are not co-transported. Thus, histidine apparently facilitates Cu uptake by competing with albumin for Cu. The results are consistent with binding of the His
2Cu(II) complex to the Cu transport protein, a ligand-exchange reaction, and transport of free ionic Cu.