Purification of proteasomes from salmonid fish sperm and their localization along sperm flagella.
We have purified two chymotrypsin-like proteases from chum salmon sperm which have no apparent acrosome structure. Both of them were high molecular mass proteases (650 kDa and 950 kDa by gel filtration) and showed 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 was 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-reacts with an antibody against the 650 kDa protease. Finally, we revealed that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-activation and molecular shape of 650 kDa salmonid protease were quite similar to those of the eukaryotic multicatalytic proteinase (proteasome), which is well known to participate in ATP-dependent degradation of ubiquitinated 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 sperm flagella is a proteasome and is a strong candidate for the factor which regulates flagellar motility in an ATP-dependent manner.
Inaba
K
K
Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Kanagawa, Japan.
Akazome
Y
Y
Morisawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012474:Salmon / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013082:Sperm Tail / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D012967:Sodium Dodecyl Sulfate; D002918:Chymotrypsin