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Evidence for the Presence of Phosphate Groups on the Two Major Proteins of the Hamster Sperm Acrosomal Matrix and Their Potential Role in Acrosomal Hydrolase Binding Activities
Author(s) -
Alvarez George,
Buchanan Teresa L,
Raychoudhury Samir,
Nagdas Subir K
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.581.5
Subject(s) - acrosin , chemistry , acrosome , phosphatase , phosphorylation , hamster , tris , capacitation , biochemistry , sperm , matrix (chemical analysis) , microbiology and biotechnology , biology , andrology , chromatography , medicine , in vitro
We previously isolated an acrosomal fraction, termed ALM, from hamster cauda epididymal spermatozoa which contains specific domains of the acrosomal matrix and an adherent detergent‐insoluble complex, termed the acrosomal lamina; which is derived from the outer acrosomal membrane. By SDS‐PAGE, the ALM fraction exhibited two major polypeptides of M r =29,000 (ALM29) and M r =20,000 (ALM 20). ALM complex binds both acrosin and N‐acetylglucosaminidase (NAGA) in a dose‐dependent manner. The present study was undertaken to examine the presence of phosphate groups on both ALM29 and ALM20 polypeptides and to identify the binding competency of dephosphorylated ALM complex to acrosomal hydrolases. Our diagonal gel electrophoresis data reveals that both ALM29 and ALM20 polypeptides are not joined by disulfide bridge(s). Both ALM29 and ALM20 polypeptides showed a reduction in size (~3 kDa) by alkaline phosphatase treatment. A sedimentation assay was employed to determine whether alkaline phosphate treated ALM possesses acrosin and NAGA binding sites. There was a ~50% reduction in binding between the dephosphorylated ALM complex and acrosin/NAGA. Our studies conclude that both ALM29 and ALM20 polypeptides are phosphorylated and that the ALM complex‐acrosomal hydrolase interactions require the phosphorylation of ALM polypeptides. Supported by NIH/NIGMS/1SC3GM096875‐01/FSU McNair and Graduate School Program.