Sea nettle (Chrysaora quinquecirrha) lethal factor: purification by recycling on m-aminophenyl boronic acid acrylic beads.
Experiments were performed to define the best isolation method for isolating Chrysaora fishing tentacle nematocyst organelles in order to minimize non-nematocyst contaminating proteins and proteases and stabilize crude nematocyst venom lethal activity. Techniques employed to disrupt the tentacles included autolysis, homogenization, or digestion using either trypsin or collagenase. Sephacryl-200 gel-filtration chromatography separated two lethal fractions. An immobilized serine protease inhibitor column, m-aminophenyl boronic acid acrylic beads, which reversibly bound one of the two lethal factors, was used in the second and third purification steps. By this means, a 105,000 mol wt. protein was purified, as judged by silver stained SDS-polyacrylamide gels. Lethal activity was inhibited by exposure to the serine protease inhibitor, L-1 chloro 3[4-tosylamido]-7-amino-2-heptanone-HCl, after purification. Although this lethal factor has some characteristics of a serine protease, it is not proteolytically active.
Long-Rowe
K O
KO
Department of Dermatology, University of Maryland, Baltimore 21201.
Burnett
J W
JW
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001897:Boronic Acids / Q000737:chemistry; D002850:Chromatography, Gel; D003064:Cnidarian Venoms / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000633:toxicity; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007928:Lethal Dose 50; D051379:Mice; D008863:Microspheres; D011480:Protease Inhibitors / Q000494:pharmacology*; D037441:Sea Nettle, East Coast; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
D000596:Amino Acids; D001897:Boronic Acids; D003064:Cnidarian Venoms; D011480:Protease Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; C028592:3-aminobenzeneboronic acid; D010450:Endopeptidases