title: Purification and Biochemical Characterization of the Hatching Enzyme from Xenopus laevis.
abstract: Using anti-GST-UVS.2 antibody and vitellin envelope (VE) as probes, the Xenopus laevis hatching enzyme (HE) was purified about 90-fold over the starting crude HE by gel-filtration and ionexchange chromatography, and its enzymatic and biochemical properties were studied. The HE has a molecular weight of 60 kD, and has high proteolytic and VE-solubilizing activities. It was very unstable during purification, and was digested easily into a 40 kD molecule, which had no VE-solubilizing activity, but still retained its proteolytic activity. The 40 kD molecule probably represents only the main protease domain in the 60 kD molecule, with two CUB repeats lost. The results on its sensitivity to EDTA and some other metal ions, combined with the occurrence of the astacin family metalloprotease-specific "HExHxxGFxHE" sequence in the deduced HE amino acid sequence, indicate that the HE is a metalloprotease. HE is very sensitive to trypsin-specific inhibitors such as leupeptin, p-APMSF, SBTI, LBTI, ovomucoid, bestatin, DFP and TLCK, which indicates that it is a trypsin-type protease. Boc-Leu-Gly-Arg-MCA had been determined to be its specific MCA-substrate.
authors.affiliation: Institute of Developmental Biology, School of Life Sciences, Shandong University, Jinan 250100, China.
authors.forename: Ting-Jun
authors.initials: TJ
authors.lastname: Fan
authors.forename: Katagiri
authors.initials: K
authors.lastname: Chiaki