Plasma membranes from rat Sertoli cells: purification and properties.
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.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011962:Receptors, FSH / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology / Q000648:ultrastructure*; D014461:Ultracentrifugation / Q000379:methods
D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone