A new membrane-associated Ca²⁺-binding protein of rat spermatogenic cells: Its purification and characterization

A Ca²⁺-binding protein of Mr=52000, estimated by SDS-PAGE, was purified to a final yield of 0.04% from rat spermatogenic cells. Purification steps included gel filtration, ammonium sulfate precipitation and HPLC. Amino acid analysis showed the content of 34% acidic residues and 15% basic residues. The isoelectric point of this protein was 4.7. Dot-blot analysis indicated that the Ca²⁺-binding protein bound 2 mol of calcium per mol of protein. This protein had two binding sites with dissociation constants of 4.8 μM and 0.2 μM. No appreciable amount of hexose was observed (<1 μg of hexose/70 ug of protein). This protein may play an important role such as the Ca²⁺-transport in the plasma membrane of spermatogenic cells.