Ovostatin, an endogenous trypsin inhibitor of sea urchin eggs: purification and characterization of ovostatin from eggs of the sea urchin, Strongylocentrotus intermedius.
A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K-21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 +/- 0.13 micrograms/ml (4.55 +/- 0.65 x 10(-8)M), 3.0 +/- 0.28 microgram/ml (1.5 +/- 0.14 x 10(-7)M), and 4.8 +/- 0.2 microgram/ml (2.4 +/- 0.1 x 10(-7)M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025-0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.
Yamada
Y
Y
Akkeshi Marine Biological Station, Faculty of Science, Hokkaido University, Japan.
Aketa
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D015387:Egg Proteins, Dietary / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005306:Fertilization; D007700:Kinetics; D008259:Macroglobulins; D008970:Molecular Weight; D010063:Ovum / Q000032:analysis*; D010179:Pancreas / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011480:Protease Inhibitors; D012617:Sea Urchins; D012965:Sodium Chloride / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D015053:Zygote / Q000032:analysis; D000511:alpha-Macroglobulins
D015387:Egg Proteins, Dietary; D008259:Macroglobulins; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D012965:Sodium Chloride; D011189:Potassium Chloride; C036828:ovomacroglobulin; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium