Isolation and Characterization of a Novel Cardiac Stimulatory and Haemolytic Protein from the Sea Anemone Urticina piscivora (Sebens and Laakso)

Ethanolic extraction of the whole bodies of the sea anemone Urticina piscivora, collected from the west coast of Canada contained a protein UpI with potent cardiac stimulatory and haemolytic activities. The protein was purified by gel chromatography, cation exchange, preparative and analytical reverse phase HPLC and shown to have a molecular size of approximately 28 kDa and pI of >9·3. The partial amino acid sequence as determined by automated Edman degradation of whole UpI is D1E-NEN5LYGPN10ENKAK15AKDLT20A GASY25LTKEA30GCTKL35QAGCT40MYQAY45N. The sequence was not identical to any of the known cardiotonic polypeptides and proteins, or cytolysins, already characterized from sea anemones. However it showed some sequence similarity to the phospholipase A2-like bungarotoxins from the snake venom of Bungarus multicintus. Even though U. piscivora belongs to the family Actiniidae, UpI lacks the initial N-terminal residues common to sea anemone proteins from this family. Using rat left atrial tissues, UpI had an ED50 of 8·1 × 10−9 M. We have shown UpI to be a potent haemolysin on erythrocytes of rat, guinea-pig, dog, pig and human. Like most sea anemone cytolysins, the haemolytic action is inhibited in a dose dependent manner by the phosholipid sphingomyelin. It was also ichthyotoxic. We thus conclude, that UpI is a novel cardiac stimulant and cytolysin by virtue of its biological action, molecular size and biochemical data. We believe we have successfully characterized for the first time a member of the third class of sea anemone cytolysin described as having phospholipase A2-like action, but for which no biochemical data are yet available.