The antihemorrhagic factor, erinacin, from the European hedgehog (Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains.
From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, alpha and beta, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine-HCl, erinacin dissociates into alpha-subunits and beta-subunit decamers. From these results the subunit assembling of erinacin has been formulated as alpha(10).2beta(10). The molecular weight of the subunits and of the beta-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.
Omori-Satoh
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Japan Science and Technology Corporation and Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand.
Yamakawa
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Y
Mebs
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D
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006364:Hedgehogs; D006470:Hemorrhage / Q000517:prevention & control*; D037102:Lectins / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097604:Ficolins
D002352:Carrier Proteins; D037102:Lectins; D008666:Metalloendopeptidases; D009124:Muscle Proteins; D011480:Protease Inhibitors; D000097604:Ficolins; C105466:erinacin protein, Erinaceus europaeus