The sperm acrosomal matrix contains a novel member of the pentaxin family of calcium-dependent binding proteins.
The sperm acrosome is a regulated secretory granule that undergoes exocytosis during fertilization. To elucidate the structural organization of the contents within the acrosome, guinea pig sperm acrosomal apical segments were isolated and mapped by two-dimensional polyacrylamide gel electrophoresis (PAGE). Although complex, the two-dimensional PAGE map was dominated by two M(r) 50,000 polypeptides (p50 and proacrosin), a M(r) 67,000 polypeptide (p67), and a M(r) 32,000 polypeptide (sp32). Proacrosin (pI > 8.0), p67, and sp32 were extracted from apical segments by 1 M NaCl. Protein p50, a relatively acidic polypeptide, was not extracted in 1 M NaCl and/or 1% Triton X-100 at 4 degrees C, but was solubilized with 6 M urea. Protein p50 was purified from the urea extract by elution from DEAE-Sephacel with 100 mM guanidine HCl and appeared homogeneous by SDS-PAGE. Antibodies to p50 were monospecific as judged by Western blot analysis. Indirect immunofluorescence indicated that p50 was restricted to the acrosomal apical segment. Incubation of apical segments at pH 7.5 in the presence of 1 mM EDTA at 37 degrees C resulted in the release of p50 into the 200,000 x g supernatant fluid, a process that was reversed by a subsequent incubation with 1.5 mM CaCl2, but not with MgCl2. The Ca(2+)-dependent reassociation of p50 with the acrosomal apical segments was reversed by the addition of 2.0 mM EGTA, indicating that p50 binding is dependent on free Ca2+ concentrations. When acrosomal matrices were purified following Triton X-100 extraction, p50 was the major component, with p67, proacrosin, and sp32 as less prominent constituents. Molecular cloning demonstrated that p50 is a unique, testis-specific member of the pentaxin family of calcium-dependent binding proteins.
Noland
T D
TD
Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232.
Friday
B B
BB
Maulit
M T
MT
Gerton
G L
GL
D000176:Acrosin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000177:Acrosome / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011506:Proteins; C022438:proacrosin; D000176:Acrosin; D002118:Calcium