Purification and characterization of protein D/E, a putative sperm-binding protein involved in fertilization.
We describe a method for the efficient purification of a 32 Kd glycoprotein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure liquid chromatography. The highly purified glycoprotein was radiolabeled with an iodinatable, cleavable, photoreactive cross-linking agent, 1-[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]-4-(-hydroxysuccini mid yl)-succinate (HAHS). The soluble radiolabeled glycoprotein was bound to washed epididymal spermatozoa in a time-dependent, saturable, and reversible manner. Scatchard analysis demonstrates that there are approximately 3,403 binding sites/spermatozoon. The binding efficiency (Kd) for spermatozoa was approximately 2.0 x 10(-10) M. The function of this glycoprotein was verified by using an in vivo artificial insemination fertilization assay. The fertility rate for control spermatozoa was approximately 53%, but the rate for spermatozoa exposed to polyclonal anti-glycoprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.
Hall
J C
JC
Department of Biochemistry, North Carolina State University Raleigh 27695-7622, USA.
Tubbs
C E
CE
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D004822:Epididymis / Q000378:metabolism*; D005260:Female; D005306:Fertilization / Q000502:physiology*; D007315:Insemination, Artificial; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013094:Spermatozoa / Q000502:physiology*; D013388:Succinimides
D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D013388:Succinimides; C047401:1-(N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl)-4-(N-hydroxysuccinimidyl)succinate