Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes

J Biochem. 1989 Feb;105(2):204-11. doi: 10.1093/oxfordjournals.jbchem.a122640.

Abstract

The hatching enzyme is an embryo-secreted enzyme(s) which digests the egg envelope, allowing the embryo to emerge at the time of hatching. The hatching enzyme of the fish, Oryzias latipes, has recently been found to consist of two kinds of proteases which may digest the inner layer of chorion (egg envelope) cooperatively [Yasumasu, S. et al. (1988) Zool. Sci. 5, 191-195]. In the present study, one of them, high choriolytic (egg envelope digesting) enzyme (HCE) was purified and some biochemical and enzymological properties were examined. The enzyme was a basic protein with a molecular weight of about 24 kDa, and exhibited choriolytic activity as well as proteolytic (caseinolytic) activity. The results of inhibitor studies and metal analyses strongly suggested that it was a zinc-protease. The purified HCE consisted of two probable isomers, HCE-1 and HCE-2. Both of them were markedly similar in amino acid composition, specific activities of choriolysis and proteolysis, and substrate specificity as determined using MCA-peptides. Moreover, they were not separable on SDS-PAGE, electrofocusing PAGE, or ultracentrifugal analysis, but were discriminated only on HPLC with a CM-300 column. Thus, the mixture of HCE-1 and HCE-2 could be regarded as almost a single enzyme, HCE. When it acted on an intact chorion, the purified HCE caused a remarkable swelling of its inner layer with concomitant release of peptides from it. Once the inner layer of chorion was swollen, the enzyme hardly digested it.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cyprinodontiformes / metabolism*
  • Densitometry
  • Electrophoresis, Polyacrylamide Gel
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Metalloendopeptidases / analysis
  • Metalloendopeptidases / isolation & purification*
  • Oryzias / metabolism*
  • Peptide Hydrolases / metabolism
  • Substrate Specificity
  • Ultracentrifugation

Substances

  • Peptide Hydrolases
  • Metalloendopeptidases
  • envelysin