A unique protease responsible for selective degradation of a yolk protein in Bombyx mori. Purification, characterization, and cleavage profile.
A yolk protein, egg-specific protein, synthesized and accumulated in the developing ovaries of Bombyx mori serves not only as the nutritive source for embryogenesis but also for the reorganization of the yolk system through limited degradation. Using the purified egg-specific protein as a substrate, a protease responsible for its limited hydrolysis was identified in embryonating eggs and purified to homogeneity. The protease had an apparent molecular mass of 30,500 with one subunit of 29,000 daltons. It hydrolyzes synthetic substrates at carbonyl bonds of Arg or Lys residues, and the hydrolysis is strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and leupeptin, suggesting that it is a trypsin-like protease. The protease shows an extremely high degree (over 2,000-fold) of specificity for egg-specific protein compared to other yolk proteins. Intact egg-specific protein is cleaved into three fragments in two steps; the first releases a 8.7-kDa peptide as an end product and a 55-kDa peptide intermediate, and in the second the intermediate is cleaved into 36- and 17.2-kDa peptides. By relating the NH2-terminal amino acid sequences of these peptides to the sequence of the intact egg-specific protein, the protease was shown to cleave first at a Lys-Asn site and secondly at Arg-Asp. Proteolytic activity abruptly appears mid-way in embryogenesis and increases steeply during completion of larval differentiation.
Indrasith
L S
LS
Laboratory of Sericultural Science, Faculty of Agriculture, Nagoya University, Japan.
Sasaki
T
T
Yamashita
O
O
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004527:Egg Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D004527:Egg Proteins; D010447:Peptide Hydrolases