Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa.
Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.
D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology; D007158:Immunologic Techniques; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology; D008854:Microscopy, Electron; D008970:Molecular Weight; D011952:Receptors, Concanavalin A / Q000032:analysis; D013077:Sperm Head / Q000648:ultrastructure; D013094:Spermatozoa / Q000648:ultrastructure*; D013347:Subcellular Fractions / Q000032:analysis
D006023:Glycoproteins; D008565:Membrane Proteins; D011952:Receptors, Concanavalin A