Sweat F W, Klee W A
J Cyclic Nucleotide Protein Phosphor Res. 1985;10(6):565-78.
Membranes from neuroblastoma X glioma NG108-15 hybrid cells were purified by equilibrium centrifugation on continuous and discontinuous gradients of sorbitol, using homogenates of cells which were pretreated with concanavalin A to increase membrane density. Adenylate cyclase was purified 24-fold in a heavy (H) membrane fraction from discontinuous gradients, opiate-stimulated guanosine-5'-triphosphatase was purified 3-fold, and opiate binding to receptors was increased 10-fold in this fraction. The relative purification of this membrane fraction is also verified by the fact that it contains a single protein (Mr = 58,000) which is covalently labeled by a reactive opiate analog (Klee, W. A., Simonds, W. F., Sweat, F. W., Burke, T. R., Jacobson, A. E., and Rice, K. C. (1982) FEBS Lett. 150, 125). The method of plasma membrane purification after cell treatment with concanavalin A (Lutton, J. K., Frederich, R. C. Jr., and Perkins, J. P. (1979) J. Biol. Chem. 254, 11181) appears generally applicable as established here with 3H-concanavalin A. Between 15 and 20% of the adenylate cyclase in whole-cell homogenates was recovered at low densities in continuous and discontinuous gradients and was only purified 2-fold above activity in the cell homogenate. There are significant differences between ligand binding, adenylate cyclase, and GTPase activities in light (L) and heavy (H) membrane fractions. GTPase activity in the L-membrane fraction was decreased from that in the cell homogenate and was not stimulated by opiates. Adenylate cyclase from L-membranes is only slightly inhibited by opiates in support of other data relating opiate inhibition to stimulation of GTPase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. 78, 4185).
用伴刀豆球蛋白A预处理细胞以增加膜密度，然后通过在连续和不连续的山梨醇梯度上进行平衡离心，从神经母细胞瘤X胶质瘤NG108 - 15杂交细胞中纯化膜。在不连续梯度的重（H）膜组分中，腺苷酸环化酶纯化了24倍，阿片类刺激的鸟苷-5'-三磷酸酶纯化了3倍，并且该组分中阿片类与受体的结合增加了10倍。该膜组分的相对纯化也通过以下事实得到验证：它含有一种单一蛋白质（Mr = 58,000），该蛋白质被一种活性阿片类类似物共价标记（克利，W.A.，西蒙兹，W.F.，斯韦特，F.W.，伯克，T.R.，雅各布森，A.E.，和赖斯，K.C.（1982年）欧洲生物化学会联合会快报150, 125）。如这里用³H-伴刀豆球蛋白A所确定的，用伴刀豆球蛋白A处理细胞后进行质膜纯化的方法似乎普遍适用。在连续和不连续梯度中，全细胞匀浆中15%至20%的腺苷酸环化酶以低密度回收，并且仅比细胞匀浆中的活性纯化了2倍。轻（L）和重（H）膜组分在配体结合、腺苷酸环化酶和GTP酶活性方面存在显著差异。L膜组分中的GTP酶活性比细胞匀浆中的降低，并且不受阿片类刺激。L膜中的腺苷酸环化酶仅受到阿片类的轻微抑制，这支持了将阿片类抑制与GTP酶刺激相关的其他数据（科斯基，G.，和克利，W.A.（1981年）美国国家科学院院刊78, 4185）。
