Characterization of a maturation-associated glycoprotein on the plasma membrane of rat caudal epididymal sperm

A Mr = 32,000 membrane glycoprotein can be uniquely labeled by galactose oxidase/[3H]sodium borohydride on rat caudal, but not caput, epididymal sperm. It has been suggested that this protein is related to a Mr = 32,000 galactose oxidase-sensitive glycoprotein present in rat caudal epididymal fluid. The tritiated membrane glycoprotein was solubilized and its hydrodynamic properties were determined by conventional gel filtration, high performance gel filtration, sedimentation rate determination in linear sucrose gradients prepared in H2O and D2O, and equilibrium isopycnic centrifugations in CsCl. The Stokes radius and sedimentation coefficient were 4.87 +/- 0.07 nm and 1.73 +/- 0.08 S, respectively. The sedimentation profile in CsCl gradients was asymmetric with a major peak occurring at a density of 1.081 g/cm3 (v = 0.92 cm3/g) and a shoulder at 1.108 g/cm3 (v = 0.90 cm3/g). The glycoprotein did not enter a 5 to 20% linear sucrose gradient prepared in D2O and could be extracted from the intact sperm into acidic chloroform:methanol solutions. These data are consistent with a protein which binds substantial amounts of detergent and/or lipid and has exposed hydrophobic regions. Two-dimensional gel electrophoresis indicated that the membrane protein exhibits charge heterogeneity, with the major components having pI values of 5.4 and 4.9. The fluid glycoprotein was monodisperse on two-dimensional gel electrophoresis having a pI of 3.8. Binding studies failed to demonstrate specific binding of the Mr = 32,000 caudal fluid glycoprotein to caput cells. Moreover, "Western blots" of electrophoretically resolved caput and caudal fluid proteins, followed by immunolabeling with antibodies raised against unfractionated caudal fluid, demonstrated the presence of a Mr = 32,000 protein in caudal fluid which was absent from caput epididymal fluid. Using the same technique, it was shown that antibodies raised against caudal fluid proteins did not cross-react with a Mr = 32,000 caudal membrane glycoprotein. Our data do not support the view that the Mr = 32,000 fluid and membrane proteins are identical.