1.biological Characterization Studies On The Crude Venom Of Spider Lycosa Singorensis 2.electrophysiological Characterization Studies Of Hainantoxin-Ⅶ And Jingzhaotoxin-Ⅳ

1. Biological characterization Studies on the Crude Venom of Spider Lycosa singorensisLycosa singorensis is a specie of spider with large body distributing in Xinjiang of China and Eastern Europe, which likes dwelling in cave. We bred the spiders gathered from Xinjiang in our lab, and collected their crude venom by stimulating with silica gel tube. We also analyzed the biological activities of the crude venom.Protein measurement indicated that the venom contained 65. 9% protein, whose molecular weight have been determined by MALDI-TOF mass spectrometry and SDS PAGE technique. It was found that crude venom had hyaluroidase, alkaline phosphatase, acid phosphatase, acetyl choline esterase, DNase with high enzymtic activities. Unexpectedly, the venom exhibited limited toxicity to mammalian after we checked it on the isolated mice Phrenic nerve diaphragm preparation experiment, the rat vas deferens preparation experiment and injected in the mice intraperitoneally. However, the crude venom at 1mg/ml caused a significant increase in the contraction of isolated toad heart preparation, and the amplitude of the contraction was augmented more than twofold. The haemolysis activity of the venom was detect on the rat blood corpuscle and its EC₅₀ value was 1.4983mg/ml. The toxic effect of crud venom were further studied on eight species of bacteria and fungi (Bacillus cereus, Corynebacterium glutamicum, Bacillus subtilis, Micrococcus luteus, Staphylococcus albus, Escherichia coli, Saccaromyces cerevieiac, Candida albicans) using antimicrobial circle method. We found that the crude venom showed strong antimicrobial activity to many kinds of Gram-positive bacteria. Finally, Venom proteins was purified preliminarily by ion exchange chromatography followed reversed phase HPLC. 2. Electrophysiological characterization Studies of Hainantoxin-Ⅶand Jingzhaotoxin-ⅣOur previous work reported that Hainantoxin-Ⅶand Jingzhaotoxin-Ⅳwere two short-chain peptides with 33 and 32 residues isolated from the venoms of Chinese tarantula Ornithoctonus hainana and Chilobrachys jzngzhao, respectively. Homology analysis suggested that both of them were stabilized by three disulfide bonds(Ⅰ-Ⅳ,Ⅱ-Ⅴ,Ⅲ-Ⅵ).But their functions remain unknown. In this study, we characterized the effect of them on sodium channels and potassium channels expressing on mammalian cardiac myocytes and insect neurons. Under whole-cell patch-clamp condition, tetrodotoxin- sensitive sodium current and transient outward potassium currents were elicited after application of depolarizing pulse on acutly isolated bollworm Helicoverpa armigeras meurons, and tetrodotoxin-resistant sodium current were induced on Sprague-Dawley rat cardiac myocytes. Exposure to hainantoxin-Ⅶat range of 1nM～1μM, both TTX-S sodium channels and transient outward potassium channels were inhibited significantly. HNTX-Ⅶat 1μM did not slow sodium channel inactivation kinetics, suggesting that it might modulate the activity of sodium channel in a similar manner to HNTX-Ⅲand HNTX-Ⅳ, two sodium channel blockers. However, different from HNTX-Ⅶ, JZTX-Ⅳevidently inhibited the inactivation kinetics of cardiac sodium channels with IC₅₀ of 0.41μM. Therefore, it is very possible that both of toxins of interest modulate the activity of sodium in a different mechanism.