Ⅰ.structure And Function Study Of Lystoxin Ⅱ:a Cytotoxic Peptide From The Wolf Spider Lycosa Singoriensis Venom Ⅱ.extablishment Of A Method Combined Oocyte Microinjection With Electrophysiological Recording

A novel cationic cytotoxic peptide with an average molecular mass of 7335.35 Da was isolated from the venom of Lycosa singoriensis, and was denoted lystoxin II. The sequence of lystoxin II was determined by Edman degradation and RACE method: KECIPKHHECTSNKHGCCRGNFFKYKCQCTTVVTQDGEQTERCFCGTPPHHK AAELVVGFGKKIF-NH2. The toxin contains 65 residues and possesses an amidated phenylalanine-residue at C-terminal. The theoretical pI of the cationic peptide is 8.69. The eight cysteins of lystoxin II are highly conserved compared with CSTX-1 and CSTX-9, indicating that lystoxin II might take the same disulfide bridge pattern as CSTX-1: C1-C4, C2-C5, C3-C8 and C6-C7 . This arrangement belongs to the inhibitor cystine knot (ICK) structural motif . Lystoxin II induced rapid leak currents in Xenopus laevis oocytes at a concentration of 50μM. Haemolytic assays of lystoxin II on human erythrocytes showed that lystoxin II induced 42% haemolysis at a concentration of 100μM. In MTT assay against Hela cells, the IC₅₀ value was 22.0μmol/L. LystoxinⅡhas no effect on Gram-positive and Gram-negative bacteria and fungi at a concentration of 50μM. PartⅡ. Establishment of a method combined oocyte microinjection with electrophysiological recordingThe capacity of the Xenopus oocytes to translate efficiently and faithfully foreign genetic information combined with its ability to assemble oligomeric receptor/channel complexes and insert them into the plasma membrane to generate excitable electrophysiological responses makes them a powerful tool for the molecular neurobiologist. Xenopus oocyte microinjection has already led to the heterologous expression of numerous ion channels, pumps, and receptors, and the cloning and characterization of the genes encoding them. Oocytes are injected with mRNAs extracted from tissues or synthesized in vitro from cloned cDNAs, after which the foreign mRNAs are translated by the oocyte's own protein-synthesizing machinery, and the products are processed and incorporated into the oocyte plasma membrane. Using this method, oocytes have been induced to acquire many known neurotransmitter receptors and voltage-activated ion channels.Once expressed in the large oocyte cells (over 1mm in diameter), these receptors and ion channels may be studied with electrophysiological and biochemical techniques in a way much easier than using neuronal cells. We establish this method combined oocyte mieroinjection with electrophysiological recording to further our insight into the effect of toxins on subtype of ion channel expressed in oocyte.