Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus.
A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD(50) of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel beta-structure, which is in good agreement with experimental data obtained from other sea anemones-actinoporins.
Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences, 690022, Vladivostok, pr. 100 let Vladivostoku 159, Russia. klyschko@piboc.dvo.ru
Elena V
EV
Klyshko
Marina P
MP
Issaeva
Margarita M
MM
Monastyrnaya
Anna P
AP
Il'yna
Konstantin V
KV
Guzev
Tatyana I
TI
Vakorina
Pavel S
PS
Dmitrenok
Tatyana A
TA
Zykova
Emma P
EP
Kozlovskaya
D000595:Amino Acid Sequence; D000818:Animals; D020029:Base Pairing; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006461:Hemolysis / Q000187:drug effects; D007928:Lethal Dose 50; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D012615:Sea Anemones; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013056:Spectrophotometry, Ultraviolet
D003603:Cytotoxins; D017931:DNA Primers