Kew D, Muffly K E, Hall P F
Biol Reprod. 1986 Dec;35(5):1235-47. doi: 10.1095/biolreprod35.5.1235.
A method is reported for preparing surface (plasma) membranes from rat Sertoli cells. The procedure is based upon homogenization in hypotonic buffer, extraction in a two-phase system, and sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by electron microscopy, enzyme markers, and functional activities associated with the membranes (binding of follicle-stimulating hormone [FSH] and production of cyclic adenosine 5'-monophosphate [cAMP]. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside of the membrane sheets. Marker enzymes for plasma membrane (5'-nucleotidase and alkaline phosphatase) showed more than 16- and 6-fold enrichment, respectively, and other enzymes showed that contamination by nuclei, mitochondria, endoplasmic reticulum, or cytosol was negligible. Binding of FSH was found to be specific, with KD 1.2 nM and the equivalent of 7500 sites per cell. This binding was enriched 20-fold compared to whole homogenate. Production of cAMP by membranes was increased by addition of FSH and by forskolin to the purified membranes in vitro.
报道了一种从大鼠支持细胞制备表面（质膜）膜的方法。该程序基于在低渗缓冲液中匀浆、在两相系统中提取以及通过两个蔗糖密度梯度沉降。纯化的膜由大片膜组成。通过电子显微镜、酶标记以及与膜相关的功能活性（促卵泡激素 [FSH] 的结合和环磷酸腺苷 [cAMP] 的产生）证明了膜的身份和纯度。电子显微镜显示膜片内部附着有小的细胞质片段。质膜的标记酶（5'-核苷酸酶和碱性磷酸酶）分别显示出超过16倍和6倍的富集，其他酶表明细胞核、线粒体、内质网或胞质溶胶的污染可忽略不计。发现FSH的结合具有特异性，KD为1.2 nM，每个细胞相当于7500个位点。与全匀浆相比，这种结合富集了20倍。在体外向纯化的膜中添加FSH和福斯高林可增加膜产生cAMP的量。
