The Biochemical Characterization of Protein DE and Its Interaction with Rat Epididymal Sperm.
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2001-03-26
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Using traditional column chromatography, Protein DE has been purified from rat epididymides. Affinity, size exclusion, and ion-exchange chromatography were utilized to purify the protein to homogeneity. Protein DE purity was demonstrated using one and two-dimensional electrophoresis. Using the purified sample, an accurate molecular mass of 27,534 Daltons was determined using electrospray-ionization mass spectrometry. After four chromatographic steps, Protein DE was efficiently separated from all detectable epididymal proteins. This report provides the first rapid and reproducible method for purifying protein DE to homogeneity.Using western blot analysis and immunofluorescence, protein D is initially detected in rat epididymal tissue and associated with sperm from the distal caput region. In contrast, when sperm were recovered from the female reproductive tract seven hours after mating, protein D was not detected by western blot, but did display faint immunofluorescence. Additionally, using photoactivatible cross-linking, a 120 KD sperm membrane protein that specifically interacts with protein D was identified. A population of membrane bound protein D was released from NaCl washed epididymal sperm when incubated in the presence of phosphatidyl-inositol specific phospholipase C. This report is the first demonstrating that both the secretion and sperm-association of protein D occur in the distal caput region of the rat epididymis. It is the only report showing western blot analysis and immunolocalization of sperm-associated protein D on sperm deposited in the female reproductive tract after mating. Additionally, this is the first report that: (a) protein D binds specifically to a 120 KD membrane protein on the surface of epididymal sperm, (b) and that protein D is anchored or associated with a protein that is anchored to the sperm plasma membrane through a glycosylphosphatidyl inositol linkage
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PhD
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Biochemistry
