Characterization of the hatching enzyme from embryos of an anuran amphibian, Rana pirica.
The culture medium in which prehatching embryos of the frog, Rana pirica, 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 solubilizing 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 of 7.6, with the apparent Km value of 250 microM at 30 degreesC. The activity was strongly inhibited by DFP and EDTA, and was accelerated by extremely low concentrations of Mg2+ and Zn2+, indicating the serine protease and metalloprotease nature of the HE. The HE was glycosylated and was present as a putative proenzyme form of 63 kDa.
Kitamura
Y
Y
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Katagiri
C
C
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000276:immunology; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011898:Ranidae / Q000196:embryology*; D012697:Serine Endopeptidases / Q000737:chemistry; D014817:Vitelline Membrane / Q000378:metabolism
D000906:Antibodies; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D009842:Oligopeptides; D010455:Peptides; C025273:prolyl-phenylalanyl-arginine-4-methylcoumaryl-7-amide; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C024344:envelysin