| Abstrakt: |
The venom of the South American spider Phoneutria nigriventer has been separated into eleven fractions by gel filtration. The neurophysiological activity of each fraction was tested by perfusion of a locust femur preparation. Fractions which gave a neurophysiological response on this perfusion were fractionated further by ion-exchange chromatography. The purity of each sub-fraction was monitored by isoelectrofocusing on polyacrylamide gels and isoelectric points determined. From one of the polypeptide-containing fractions, a pure, almost neutral polypeptide was isolated and shown to have a molecular weight between 5500 and 5900. The amino acid composition of the pure polypeptide was: Ala6,7 Arg2,3 Asx3 Cys8 Glx3 Gly4 Ile4 Leu1 Lys5,6 Phe2 Ser5 Thr3 Trp1 Tyr2 Val2. This polypeptide elicited the greatest neurophysiological activity of all fractions tested. When the polypeptide (2 X 10(-7) M) was perfused through the locust femur preparation, action potentials were generated along the length of the axons in the crural nerve, resulting in very rapid and uncontrolled twitching of the skeletal muscles. At higher concentrations, the crural nerve discharged repetitively, both spontaneously and in response to a single electrical stimulus. The other polypeptide fractions, although less pure, had neurophysiological responses similar to those observed with the pure polypeptide; the effects of some fractions could be reversed. Further fractions of low molecular weight were purified by thin-layer or paper chromatography to give two pure components that are probably nucleosides or nucleotides. After the initial gel filtration of the total venom had separated the high molecular weight proteinases from the polypeptides, all of the polypeptides retained their neurophysiological activity in solution for several days. In the presence of the proteinases, the polypeptides were inactivated in solution in a few hours at room temperature. |
