Tezapsidis N, Parish D C
Unit of Metabolic Medicine, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London.
Gen Comp Endocrinol. 1994 Aug;95(2):248-58. doi: 10.1006/gcen.1994.1122.
Preparations containing ovine chromaffin granules and lysosomes were obtained by differential centrifugation and applied to density gradients. In isoosmolar linear Metrizamide gradients the granules had a lower density than the major portion of the lysosomes (1.05 compared to 1.15); however, in hyperosmolar linear Metrizamide gradients the granule density increased and they migrated close to the lysosomes. The granules separated into two bands on a discontinuous isoosmolar Metrizamide gradient; however, these two bands were similar in terms of granule and lysosomal markers. On a discontinuous hyperosmolar sucrose gradient the granules were more dense than the lysosomes, the reverse of the situation on the Metrizamide gradient. Separation on a discontinuous isoosmolar Metrizamide gradient followed by a 1.8 M sucrose cushion provided a 54-fold purification of granules from lysosomes and similar separations from other subcellular markers. This procedure also provided a 37-fold purification of bovine granules from lysosomes. It was demonstrated that thimet oligopeptidase (EC 3.4.24.15) occurred in the adrenal medulla but is not principally located in the chromaffin granule.
通过差速离心获得含有绵羊嗜铬颗粒和溶酶体的制剂，并将其应用于密度梯度。在等渗线性泛影葡胺梯度中，颗粒的密度低于溶酶体的主要部分（分别为1.05和1.15）；然而，在高渗线性泛影葡胺梯度中，颗粒密度增加，它们迁移至靠近溶酶体的位置。在不连续等渗泛影葡胺梯度上，颗粒分离成两条带；然而，就颗粒和溶酶体标志物而言，这两条带相似。在不连续高渗蔗糖梯度上，颗粒比溶酶体密度更大，这与泛影葡胺梯度的情况相反。在不连续等渗泛影葡胺梯度上进行分离，随后经1.8 M蔗糖垫层处理，可使颗粒相对于溶酶体得到54倍的纯化，相对于其他亚细胞标志物也有类似程度的分离。该方法还可使牛颗粒相对于溶酶体得到37倍的纯化。已证明硫醇寡肽酶（EC 3.4.24.15）存在于肾上腺髓质中，但主要并不位于嗜铬颗粒中。
