Deupree J D, Weaver J A, Downs D A
Biochim Biophys Acta. 1982 Feb 25;714(3):471-8. doi: 10.1016/0304-4165(82)90156-8.
Studies on the mechanism of catecholamine transport into chromaffin granules is complicated by the release of endogenous catecholamines. To overcome this problem chromaffin granule ghosts have been prepared by many investigators by osmotic lysis of the granules which results in a loss of over 90% of the endogenous catecholamine. However, in the studies reported here, the resulting ghosts still contained 36 +/- 3.9 nmol epinephrine/mg of protein if they were lysed by passage through a Sephadex G-50 column preequilibrated with hypoosmotic media. This residual catecholamine was found to slowly diffuse out of the ghosts in a temperature-dependent process at a rate sufficient to interfere with kinetic analysis of catecholamine transport. Attempts to remove the endogenous catecholamine from the ghosts indicated that most of it could not be removed by further osmotic shock of freeze-thaw treatments, but that over 85% of it was released from the granules by incubating them at 30 degree C for 90 min or by dialysis with a 35 and 86% loss of rate of catecholamine transport into the ghosts, respectively. If the endogenous catecholamine was removed from chromaffin granule ghosts by preincubating them for 90 min at 30 degree C, the resulting ghosts transported catecholamine with a linear Lineweaver-Burk plot indicating a Km of 12 +/- 2 microM. In addition, the resulting ghosts did not leak catecholamines over a 10 min period at 30 degree C, and the transport of catecholamines was blocked by reserpine and enhanced with increasing pH from 6.0 to 8.5.
内源性儿茶酚胺的释放使得对儿茶酚胺转运至嗜铬颗粒机制的研究变得复杂。为克服这一问题，许多研究者通过对颗粒进行渗透裂解制备了嗜铬颗粒空泡，这导致超过90%的内源性儿茶酚胺流失。然而，在本文报道的研究中，如果通过用低渗介质预平衡的葡聚糖G - 50柱裂解嗜铬颗粒空泡，所得到的空泡每毫克蛋白质仍含有36±3.9纳摩尔肾上腺素。发现这种残留的儿茶酚胺以温度依赖的过程缓慢地从空泡中扩散出来，其速率足以干扰儿茶酚胺转运的动力学分析。从空泡中去除内源性儿茶酚胺的尝试表明，通过进一步的渗透休克或冻融处理，大部分内源性儿茶酚胺无法被去除，但通过在30℃孵育90分钟或将其透析，分别有超过85%的内源性儿茶酚胺从颗粒中释放出来，儿茶酚胺向空泡转运的速率分别损失35%和86%。如果通过在30℃预孵育90分钟从嗜铬颗粒空泡中去除内源性儿茶酚胺，所得到的空泡转运儿茶酚胺时呈现线性的Lineweaver - Burk图，表明Km为12±2微摩尔。此外，所得到的空泡在30℃下10分钟内不会泄漏儿茶酚胺，并且儿茶酚胺的转运被利血平阻断，并且随着pH从6.0增加到8.5而增强。
