CHARACTERIZATION OF THE HATCHING ENZYME FROM EMBRYOS OF AN ANURAN AMPHIBIAN, RANA-PIRICA

The culture medium in which prehatching embryos of the frog, Rana piri ca, were cultured (hatching medium) solubilized the vitelline coat (VC ) of unfertilized eggs and contained molecules reactive to antibodies (anti-UVS.2) against the Xenopus hatching enzyme (HE). The hydrolyzing activity of the hatching medium against Pro-Phe-Arg-MCA was inhibited dose-dependently by the same antibodies. Using anti-UVS.2 as a probe, we purified two distinct 56 kDa molecules exhibiting Pro-Phe-Arg-MCA hydrolyzing activity. These 56 kDa molecules, which were separable on anion exchange chromatography, were the same with respect to VC solubi lizing activity and a substrate specificity for various MCA-peptides, and they were regarded as charge isomers that function as the HE. The hydrolyzing activity against Pro-Phe-Arg-MCA of HE was optimal at pH o f 7.6, with the apparent K-m value of 250 mu M at 30 degrees C. The ac tivity was strongly inhibited by DFP and EDTA, and was accelerated by extremely low concentrations of Mg2+ and Zn2+, indicating the serine p rotease and metalloprotease nature of the HE. The HE was glycosylated and was present as a putative proenzyme form of 63 kDa. (C) 1998 Elsev ier Science B.V. All rights reserved.