Barnea A, Katz B M
Department of Obstetrics and Gynecology, University of Texas, Southwestern Medical Center, Dallas 75235.
J Inorg Biochem. 1990 Sep;40(1):81-93. doi: 10.1016/0162-0134(90)80041-u.
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.
先前的研究表明，67Cu与组氨酸（His）的络合促进下丘脑切片对67Cu的摄取，而浓度比Cu(His)2高1000倍的组氨酸会抑制67Cu的摄取（D. E. Hartter和A. Barnea，《生物化学杂志》263, 799 - 805 (1988)）。我们探讨了以下问题：在Cu2+摄取过程中，Cu(His)2络合物是否会发生解离？如果会，解离是决定摄取的唯一因素吗？将大鼠下丘脑切片与67Cu(3H - His)2一起孵育，并评估67Cu和3H - His摄取的动力学曲线。67Cu摄取在长达60分钟内呈线性，Vo与S [0.1 - 160 microM Cu(His)2]呈S形，Lineweaver - Burk图呈非线性，Scatchard图呈钟形，Hill图有多个斜率。相比之下，3H - His摄取在长达30分钟内呈线性，Vo与S呈双相，Lineweaver - Burk图呈线性，Scatchard图呈双相，Hill图有单个斜率。保持[67Cu]不变并增加[3H - His]会导致67Cu摄取呈剂量依赖性抑制，而3H - His摄取不受抑制。用苯丙氨酸（Phe）或赖氨酸（Lys）取代络合物中的组氨酸会导致67Cu摄取的Vo与S明显右移，并且在S小于40 microM时，只有组氨酸相对于离子67Cu2+促进67Cu摄取。然而，3H - His、3H - Phe和3H - Lys摄取的Vo与S是可叠加的，表明络合物的解离程度相当。总之，我们证明，虽然Cu2+的络合对于下丘脑组织摄取67Cu至关重要，但67Cu和3H - His通过不同的过程被摄取，这意味着络合物在膜水平发生解离。此外，即使发生解离，它也不是决定下丘脑组织摄取Cu2+的唯一因素。有人提出，Cu络合物的物理化学性质是决定脑组织摄取Cu的一个重要因素。
