| Abstract
|
:
|
Eleven sea anemones species (Anthopleura elegantissima, A. xanthogrammica, Corynactis californica, Epiactis prolifera, Metridium senile, Pachycerianthus fimbratus, Stomphia didemon, Urticina coriacea, U. crassicornis, U. lofotensis and U. piscivora), collected from the west coast of Canada, were screened for cardiotonic, cytolytic, cytotoxic and antifungal activities. Extracts from nine species elicited cardiotonic activity on rat atria. While P. fimbratus was inactive, E. prolifera was cardiotoxic. Eight extracts were hemolytic on erythrocytes of rat, guinea pig and dog; six exhibited cytotoxic activity to KB, L1210 and HEL 299 cell lines. Extracts from all anemone species were devoid of antifungal activity against Aspergillus niger PLM 1140 and Candida albicans ATCC 14053. From extracts of U. piscivora, three cardiotonic proteins (UpI, UpII and UpIII) were purified using a combination of chromatographic methods. UpI is a basic protein, (pI > 9.3), of molecular weight 28 kDa, and N-terminal amino acid sequence of usd\rm D\sp1ENEN\sp5LYGPN\spENKAK\spAKDLT\sp{20}AGASY\sp{25}LT KEA\sp{30}GCT KL\sp{35}QAGCT\sp{40}MYQAY\sp{45}Nusd. It elicited potent cardiac stimulatory activity on rat left atria (EC{50}: usd8.1\times 10\sp{-9}usd M) comparable to isoproterenol (EC{50}: usd3\times 10\sp{-9}usd M). Although different from known anemone proteins, UpI showed some sequence similarity to the bungarotoxins from Bungarus multicintus. UpII and UpIII (pI = 7.2 and 7.6, respectively) appear to be variants of each other with similar molecular weights of 19 and 40 kDa in reducing and non-reducing conditions, respectively. Their N-terminal amino acid sequences were usd\rm A\sp1TDKW\sp5NDCG S\spVTALC\spEQKGF\sp{20}NKATC\sp{25}usd (UpII), and usd\rm D\sp1DDWD\sp5EGCHV\spTALLEGQQGR\sp{20}NK AAC\sp{25}usd (UpIII). UpII (ED{50}: usd1\times 10\sp{-7}usd M) was more potent on rat left atria than UpIII (ED{50}: usd7\times 10\sp{-6}usd M). UpI was hemolytic to erythrocytes of rat, guinea pig, dog, pig and human. Using scanning electron microscopy, it was shown to cause structural damage to the membranes of rat and guinea pig erythrocytes. Hemolysis was inhibited by sphingomyelin. UpII and UpIII were not hemolytic. Pathophysiological investigations of all proteins revealed only UpI to be toxic, causing hypotension, respiratory and circulatory failure in anesthetized rats, similar to the crude extract from U. piscivora. Autopsy revealed morphological damage to the skin, lungs, liver and kidney, but not the heart and brain. All three proteins were devoid of antifungal activity. While UpI appears to belong to the class of toxins with phospholipase A activity, UpII and UpIII appear to be a new class of cardiotonic proteins.
|