PURIFICATION OF PROTEASOMES FROM SALMONID FISH SPERM AND THEIR LOCALIZATION ALONG SPERM FLAGELLA

We have purified two chymotrypsin-like proteases from chum salmon sper m which have no apparent acrosome structure. Both of them were high mo lecular mass proteases (650 kDa and 950 kDa by gel filtration) and sho wed not only chymotrypsin-like activity but also trypsin-like activity . The 650 kDa protease was composed of at least eight or nine kinds of polypeptide with molecular masses ranging from 20 kDa to 30 kDa and w as highly activated by low concentrations of SDS. Electron microscopy revealed that the 650 kDa protease was a ring-shaped particle. The 950 kDa protease was shown to contain at least one component that cross-r eacts with an antibody against the 650 kDa protease. Finally, we revea led that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-acti vation and molecular shape of 650 kDa salmonid protease were quite sim ilar to those of the eukaryotic multicatalytic proteinase (proteasome) , which is well known to participate in ATP-dependent degradation of u biquitinated proteins; and, furthermore, the motility of demembranated sperm of salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. Thus, the protease located in salmonid fish sper m flagella is a proteasome and is a strong candidate for the factor wh ich regulates flagellar motility in an ATP-dependent manner.