Svoboda P, Kvapil P, Insel P A, Ransnäs L A
Institute of Physiology, Czech Academy of Sciences, Prague.
Eur J Biochem. 1992 Sep 15;208(3):693-8. doi: 10.1111/j.1432-1033.1992.tb17236.x.
We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the alpha subunit of Gs (Gs alpha) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900-7903], three membrane-bound forms of Gs alpha were identified through rate-zonal centrifugation in sucrose density gradients, Gs alpha-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gs alpha in the high-density portion of the gradient (1.13-1.16 g/cm3) is identical with that of beta-adrenergic-receptor binding, Na/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09-1.11 g/cm3), contains a much lower total amount of Gs alpha and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH-cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of Gs alpha located at the top of the gradient (1.06-1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of Gs alpha is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100,000 x g proves its particulate (membrane) character. The light membrane form of Gs alpha subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of Gs alpha. Overall, our data indicates that a substantial portion of Gs alpha is localized in membrane pools other than plasma membrane.
我们报告，刺激性鸟嘌呤核苷酸结合调节蛋白（Gs）在S49淋巴瘤细胞中存在区室化现象。除了先前报道的Gsα亚基的胞质形式[Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900 - 7903]，通过在蔗糖密度梯度中进行速率区带离心、Gsα特异性抗肽血清和腺苷酸环化酶互补测定，鉴定出了三种膜结合形式的Gsα。梯度高密度部分（1.13 - 1.16 g/cm³）中第一部分Gsα的沉降图谱与β - 肾上腺素能受体结合、Na/K - ATP酶和腺苷酸环化酶活性的沉降图谱相同，因此可能被鉴定为质膜片段。在梯度中间部分（1.09 - 1.11 g/cm³）回收的第二部分，Gsα的总量要低得多，并且与内质网（微粒体）酶标记物NADPH - 细胞色素c还原酶和葡萄糖 - 6 - 磷酸酶相关。位于梯度顶部（1.06 - 1.08 g/cm³）的第三部分Gsα的身份未知。高尔基体标记物UDP半乳糖：N - 乙酰葡糖胺糖基转移酶在该区域部分回收；然而，这种酶在梯度的高密度部分也存在。完全没有特异性腺苷酸环化酶和Na/K - ATP酶活性表明，这种低密度（轻）膜形式的Gsα与任何质膜片段都不同。此外，在100,000 x g下的沉降证明了其颗粒（膜）性质。在使用缺乏Gsα的cyc - 膜进行的腺苷酸环化酶互补测定中，Gsα亚基的轻膜形式具有功能活性。总体而言，我们的数据表明，相当一部分Gsα定位于质膜以外的膜部分。
