| Abstract: | A species-specific factor capable of disersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassisina. It does not exert its effect on the vitelline layer. The purification has been accomlished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatograhy on heparin-Seharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrohoresis in the presence or absence of β-mercatoethanol, estimated molecular weight being about 140,000. The jelly dispersion by the present factor is activated by CaCl2, and inhibited by KCl, MnCl2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate, Inorganic sulfated such as (NH4)2SO4 and Na2SO4 have no effect on jelly dispersion. This factor is heat-labile, its activity in 30 min at 50°C. The present factor is found also in the seminal Plasma, and released from sperm themselves by treatment with Triton X-100 .These results suggest that this factor is loosely bound to the serm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly disersing factor. Only trypsin and α chymotrysin among commercial enzymes tested dispersing activity is inhibited neither by trypsin inhibitors such as N-α-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-1-tosylamide-2 pheny-ethylcholoromethyl ketone and chymostatin Participation of trysin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely. |
