Semen from the sea urchin Strongylocentrotus purpuratus contains sperm and a small volume (1%) of phagocytes, which often contain degraded sperm. A 53,000-Da
esterase in the semen is inhibited by diisopropyl
fluorophosphate, but not by soybean
trypsin inhibitor (
STI). Differential centrifugation experiments now reveal that 70% of the
esterase activity (formerly described as a sperm
protease precursor; Levine and Walsh, 1980) is associated with the phagocytes, which sediment more rapidly than the sperm. The 53K
esterase is also present in spawned ovaries and testes. However, as previously reported, the sperm do contain an
STI-inhibitable
protease as shown by the digestion of [14C]
lysozyme. Intact sperm exhibit
STI-inhibitable hydrolytic activity toward N-alpha-[3H]
benzoyl-L-arginine ethyl ester [3H]
BAEE), but crude homogenates do not until they are extracted at pH 2.5 and fractionated by ion exchange chromatography. Although not obtained in pure form, the
protease activity appears to migrate with a molecular weight of 20,000 (gel filtration). The
protease and the
esterase differ markedly in
acid stability. After preincubation at pH 2.5 the
protease still hydrolyzes [3H]
BAEE, while the
esterase is irreversibly inactivated. This last observation may explain an earlier interpretation (A. E. Levine and K. A. Walsh, 1980, J. Biol. Chem. 255, 4814-4820) that the 53K
enzyme dissociated at pH 2.5 into two unequal subunits, one of which was the active
protease. Since it has been shown that the contaminating phagocytes contribute most of the
esterase activity of the semen, the occurrence of even a small number of nonsperm cells cannot be ignored in future investigations of sperm
enzymes.