Abstract
Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three samples in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / chemistry
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Animals
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Blotting, Western
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Cyclic AMP / chemistry
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Cyclic AMP-Dependent Protein Kinases / chemistry
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Dextrans / pharmacology
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Electrophoresis, Gel, Two-Dimensional / methods*
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Electrophoresis, Polyacrylamide Gel
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Epididymis / metabolism
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Fluorescent Dyes / pharmacology
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Hydrogen-Ion Concentration
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Male
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Mass Spectrometry
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Mitochondria / metabolism
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Phosphorylation
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Protein Processing, Post-Translational*
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Proton-Translocating ATPases / chemistry*
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Rats
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Rats, Wistar
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Rhodamines / pharmacology
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Serine / chemistry
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Spermatozoa / chemistry
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Spermatozoa / metabolism*
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Trypsin / chemistry
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Trypsin / pharmacology
Substances
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Dextrans
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Fluorescent Dyes
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Fluoro-Ruby
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Rhodamines
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Serine
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Cyclic AMP
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Cyclic AMP-Dependent Protein Kinases
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Trypsin
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Adenosine Triphosphatases
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Proton-Translocating ATPases