By using Artemia chorion as a specific substrate, the hatching enzyme from Anemia salina (AHE) was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymaticaily in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and often contained 73.3 kDa mol-ecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 40℃. The K_m value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophe-nyl methane sulfonyl fluoride hydrochioride, greatly inhibited by N-tosyl-L-lysyl chloromethyi ketone, phenyl-methanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyI-L-phenylalanyl chioromethyl ketone, leupeptin, N-ethyimaleimide, and iodoacetamide, and not inhibited by chymostatin and bes-tatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn~(2+). Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn~(2+), it is indicated that AHE might be also a kind of Zn-metalloprotease.
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