Abstract
We characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system. Two ligand-dependent saturable processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process. Vo versus [S] for the high affinity process was a hyperbolic function having an apparent Km and Vmax of 6 microM copper and 23 pmol/min/mg protein, respectively. Vo versus [S] for the low affinity process was a sigmoidal function having an "apparent Km" (So5) and maximal velocity at saturating [S] of 40 microM copper and 425 pmol/min/mg protein, respectively. The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation; (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand; (c) a requirement for thermic but not metabolic energy. In spite of these similarities, a 50- or 1000-fold molar excess of ligand (histidine) inhibited 67Cu accumulation by the low affinity process by 60 and 85%, respectively, whereas excess histidine facilitated 67Cu accumulation by the high affinity process by 1.6-4-fold. These results are consistent with 1) a carrier-mediated facilitated diffusion, analogous to that of neutral amino acids, as a means of transporting complexed copper into brain tissue, and 2) the existence of two distinct carrier sites interacting in a positive cooperative manner: a high and a low affinity site.
Highlights
From the Departmentsof Obstetrics and Gynecologyand Physiology, Cecil H. and Ida Green Centerfor Reproductiue Biology Sciences, The University of Texas HealthScience Center, D a l h, Texas 75235
We considered the possibility that 67Cuaccumulated by the hypothalamic tissue by two processes, a high affinity process operating at 2.5-5 ~ L M67Cu.Since we found that a 1000-fold excess histidineinhibited T u accumulation when 20 p~ but not when 0.2 p~ 6 7 C ~ H i sw2 as used (Fig. 2), we reasoned that excess histidine would facilitate accumulation of 67Cu bythe high affinity process and suppress that of the low affinity process
We demonstrate that hypothalamic tissues, incubated under in vitro conditions, accumulate T u by two ligand-dependent saturable processes: a high affinity, low capacity process exhibiting an apparent K, of 6 p~ copper and V, of 23 pmol/min/mg protein; and a low affinity, high of 67Cuby Brain Tissue capacity process exhibiting an “apparent K,” (So,so)f about for aminoacids
Summary
Since the circulatinglevels of copper are in the rangoef 20 counts/min values were corrected for the 61-h half-life decay of 67Cu. pM [7, 9, 10] and since the potential concentrationof copper. Tissueslices were incubated with0.2 p~ fi7C~Hisfo2r as a function of the molar ratio of copper:histidine reaching various periods of time (5-60 min), and the tissue was a peak value at a ratio of 1:2,000. Since the optimal copper- 20 p~ copper, a molarratio of 1:lor 12.The following ligands histidine complex is a t a ratio of 1:2 [28, 29, 34], hypothalami were examined histidine, histamine, threoninec,ysteine, glywere incubated with either ionic 67Cu(67CuCl,)or complexed cine, Gly-His-Lys, GSH, anbdovine serum albumin. To assess whether the ligand (histidine) competewsith the GSH may reduce the Cu(I1) and sinceexcess albumin inhibits copper complex, hypothalami were incubated with 0.2 or 20 accumulation of 67Cu by livercells[21]. Hypothalami were incubated for 30 min in the presence of 0.2 p~ 6 7 Cc~omplexed to either histidine, GSH, or bovineserum albumin at the molar ratios shown in the table
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
