Purification and characterization of proteases responsible for vitellin degradation of the silkworm, Bombyx mori

Abstract

Hydrolytic activity of benzoyl- dl-arginine -p- nitroanilide (BApNA) by crude egg extracts exhibited two peaks during the latter period of embryogenesis of the silkworm, Bombyx mori. The first peak appeared at the time of marked degradation of vitellin, and consisted of two proteases. The two proteases were purified from day 8 eggs to homogeneity by DEAE-cellulose, Sephadex G-75 and native-gel electrophoresis. One enzyme had a molecular mass of 30,000 Da and the other 24,000 Da. The NH 2-terminal amino acid sequences were different from one another and only the 30 k-protease showed the sequence common to the trypsin family. Both enzymes showed similar properties in K m values and optimum pH, but different temperature dependence. Inhibition spectrum and substrate specificity suggest that these enzymes belong to the trypsin-like serine protease family. The purified proteases cleaved two yolk proteins, vitellin and egg-specific protein, into small peptides through limited hydrolysis, but could not attack the 30 kDa proteins which are the second major yolk proteins of silkworms. Degradation of vitellin was characterized by the preferential hydrolysis of a 178 kDa large subunit. From the biochemical properties and developmental changes, these two proteases appear to be responsible for the degradation of vitellin in eggs undergoing embryogenesis.