Properties of the hatching enzyme from Xenopus laevis.
Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.
Fan
T J
TJ
Department of Marine Biology, College of Marine Life Sciences, Ocean University of Qingdao, China. tjfan@ouqd.edu.cn
Katagiri
C
C
D000818:Animals; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D016622:Silver Staining; D012995:Solubility; D013379:Substrate Specificity; D014817:Vitelline Membrane / Q000378:metabolism; D014982:Xenopus laevis; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D002364:Caseins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; C002087:N,N-dimethylcasein; D008666:Metalloendopeptidases; C024344:envelysin; D015032:Zinc