Uptake of 67copper complexed to 3H-histidine by brain hypothalamic slices: evidence that dissociation of the complex is not the only factor determining 67copper uptake

Abstract

It was previously shown that complexation of 67Cu with His facilitates 67Cu uptake by hypothalamic slices and that His, in a concentration that is 1000-fold greater than Cu(His)2, inhibits 67Cu uptake (D. E. Hartter and A. Barnea, J. Biol. Chem. 263, 799-805 (1988)). We addressed the question: Does dissociation of the Cu(His)2 complex occur during the process of Cu2+ uptake and if so, is dissociation the only factor determining uptake? Rat hypothalamic slices were incubated with 67Cu(3H-His)2 and the kinetic profiles of 67Cu and 3H-His uptake were evaluated. 67Cu uptake was linear for up to 60 min, Vo vs S [0.1-160 microM Cu(His)2] was sigmoidal, Lineweaver-Burk plot was non-linear, Scatchard plot was bell-shaped, and Hill plot had multiple slopes. In contrast, 3H-His uptake was linear for up to 30 min, Vo vs S was biphasic, Lineweaver-Burk plot was linear, Scatchard plot was biphasic, and Hill plot had a single slope. Keeping [67Cu] constant and increasing [3H-His] resulted in a dose-dependent inhibition of 67Cu uptake which was not accompanied by an inhibition of 3H-His uptake. Substituting His in the complex with Phe or Lys resulted in a marked shift to the right in Vo vs S for 67Cu uptake and at S less than 40 microM, only His facilitated 67Cu uptake relative to ionic 67Cu2+. However, Vo vs S for 3H-His, 3H-Phe, and 3H-Lys uptake were superimposeable, indicating comparable dissociation of the complexes. In summary, we demonstrate that, although complexation of Cu2+ is essential for 67Cu uptake by hypothalamic tissue, 67Cu and 3H-His are taken up by distinct processes, which implies dissociation of the complex at the level of the membrane. Moreover, even though dissociation occurs, it is not the only factor that determines Cu2+ uptake by the hypothalamic tissue. It is suggested that the physicochemical properties of the Cu complex is an important factor determining Cu uptake by brain tissue.