Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa.
The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.
D000177:Acrosome / Q000032:analysis* / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008297:Male; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification; D013094:Spermatozoa / Q000032:analysis*
D046911:Macromolecular Substances; D008565:Membrane Proteins; D010455:Peptides