Identification of the rat epididymis-secreted 4E9 antigen as protein E: further biochemical characterization of the highly homologous epididymal secretory proteins D and E.
Epididymis-secreted proteins D and E have been purified to homogeneity and partially characterized, and it is shown that monoclonal antibody (MAb) 4E9 (raised against a detergent extract of rat caudal epididymal sperm [Moore et al., 1994: Mol Reprod Dev 37(2):181-194]) recognizes protein E, but not protein D. The molecular weight of protein D (approximately 30 kD) is approximately 2 kD lower than protein E (approximately 32 kD). The NH2-terminus of each protein is blocked; however, microsequencing of internal peptides confirms earlier reports of significant sequence identity between the two proteins. High performance liquid chromatography tryptic peptide mapping showed peak differences between the two proteins, but it was not possible to obtain amino acid sequence in the peaks that were different. The epitope for MAb 4E9 was localized in the blocked NH2-terminus-CNBr peptide derived from protein E. The epitope was destroyed by protease treatment of protein E. Removal of N-linked oligosaccharides did not destroy the epitope for MAb 4E9 and did not affect the molecular weight difference between the proteins.
Xu
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Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis 55455, USA.
Hamilton
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DW
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003488:Cyanogen Bromide; D030501:Epididymal Secretory Proteins; D004822:Epididymis / Q000737:chemistry*; D008297:Male; D008667:Metalloproteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D017207:Rats, Sprague-Dawley; D013094:Spermatozoa / Q000276:immunology; D013735:Testicular Hormones / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D014357:Trypsin
D000911:Antibodies, Monoclonal; D030501:Epididymal Secretory Proteins; D008667:Metalloproteins; D009844:Oligosaccharides; D013735:Testicular Hormones; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003488:Cyanogen Bromide