Kobold G, Langer R, Burger A
Naunyn Schmiedebergs Arch Pharmacol. 1985 Nov;331(2-3):209-19. doi: 10.1007/BF00634240.
By osmotic lysis in the presence of urea ghosts (60-100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4-6 mumol catecholamine/mg prot.) of the bovine medulla. In the presence of 1-300 mumol/l 3H-catecholamine and ATP-Mg2+, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (delta p). The same uptake was found by 3H-counting or by fluorimetric measurements. At various pH-values (pH 6.2-8.2) the Km and Vmax of the ATP-stimulated rate of uptake of 3H-catecholamine into ghosts was determined (at 30 degrees C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (delta pH = pHout - pHin) and the electrical potential difference (delta psi) were determined to calculate delta p under conditions of 3H-catecholamine uptake. When the pHout was increased (pH 6.2, 7.4, 8.2), the apparent Km of uptake decreased (50, 5, 1-2 mumol/l), showing a linear relation between pH and logarithm of Km. The Km was calculated for the uncharged catecholamine (with pK1 = 8.8 and pK2 = 10.0); it was nearly pH-independent and amounted to about 0.2 mumol/l. The Vmax declined only in the extreme pH-range. Between pH 6.6 and 7.8 Vmax and delta p showed a slight increase from 16 to 20 nmoles/(mg prot. X min) and from 110 to 140 mV, resp. In the same pH-range the pHin inside ghosts increased from pH 5.2 to 5.7, whereas delta psi was constant (30 mV). At constant pHout (= 7.3) ammonium (0-30 mmol/l) caused an increase of pHin from 5.5 to 6.6. The increase of pHin was accompanied by an increase of Km from 5 to 20 mumol/l 3H-catecholamine and by a decrease of both Vmax and delta p from 20 to 5 nmoles/(mg prot. X min) and from 123 to 85 mV, respectively. From the dependence of the Km of uptake on pHout is concluded that the uncharged species of catecholamine is transported, whereas the dependence of Km on pHin suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.
通过在尿素存在下进行渗透裂解，从牛髓质的嗜铬颗粒（4 - 6 μmol儿茶酚胺/ mg蛋白）制备出空泡（60 - 100 nmol儿茶酚胺/ mg蛋白）。在1 - 300 μmol/l 3H - 儿茶酚胺和ATP - Mg2 +存在的情况下，空泡表现出儿茶酚胺的净摄取。该净摄取对利血平或降低颗粒膜质子电化学势差（Δp）的试剂（解偶联剂和铵）敏感。通过3H计数或荧光测量得到相同的摄取情况。在不同pH值（pH 6.2 - 8.2）下（30℃）测定了ATP刺激的3H - 儿茶酚胺摄取到空泡中的速率的Km和Vmax，以确定作为颗粒载体底物的儿茶酚胺种类（质子化、不带电荷或阴离子形式）。在3H - 儿茶酚胺摄取条件下测定pH差值（ΔpH = pH外 - pH内）和电势差（Δψ）以计算Δp。当pH外升高（pH 6.2、7.4、8.2）时，摄取的表观Km降低（50、5、1 - 2 μmol/l），显示出pH与Km对数之间的线性关系。计算了不带电荷的儿茶酚胺的Km（pK1 = 8.8，pK2 = 10.0）；它几乎与pH无关，约为0.2 μmol/l。Vmax仅在极端pH范围内下降。在pH 6.6至7.8之间，Vmax和Δp分别从16增加到20 nmol/（mg蛋白×min）和从110增加到140 mV。在相同pH范围内，空泡内的pH内从pH 5.2增加到5.7，而Δψ保持恒定（30 mV）。在恒定pH外（= 7.3）时，铵（0 - 30 mmol/l）使pH内从5.5增加到6.6。pH内的增加伴随着Km从5增加到20 μmol/l 3H - 儿茶酚胺，同时Vmax和Δp分别从20下降到5 nmol/（mg蛋白×min）和从123下降到85 mV。从摄取的Km对pH外的依赖性得出结论，儿茶酚胺的不带电荷形式被转运，而Km对pH内的依赖性表明儿茶酚胺 - 载体复合物跨颗粒膜的转运不是儿茶酚胺摄取的限速步骤。
