Immunological evidence for a P2 protamine precursor in mature rat sperm.
High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.
Stanker
L H
LH
Lawrence Livermore National Laboratory, University of California, Livermore.
McKeown
C
C
Balhorn
R
R
Lee
C
C
Mazrimas
J
J
Goralka
M
M
Wyrobek
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008297:Male; D011479:Protamines / Q000276:immunology / Q000378:metabolism*; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D013045:Species Specificity; D013094:Spermatozoa / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011479:Protamines; D011498:Protein Precursors; C069925:protamine P2 precursor