Isolation of rhodopsin by the combined action of cardiotoxin and phospholipase A2 on rod outer segment membranes.

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom VII4 cardiotoxin in rod outer segment membrane preparations. The extent of the morphological changes depend on the purity of the cardiotoxin. Pure cardiotoxin had no detectable effect upon the preparation, but, when contaminated with venom phospholipase A2, led to a rapid disintegration of the membrane vesicles. With trace amounts (up to about 0.5% of the cardiotoxin) of phospholipase A2, the membrane vesicles disintegrated into smooth lamellae and particles in solution. These two components were separated by centrifugation. The pellet, which showed the presence of smooth lamellae and aggregated particles, was composed of unbleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. The supernatant, which showed only the presence of dispersed particles, was composed of unbleached rhodopsin, lysolipids and cardiotoxin. With cardiotoxin contained larger amounts of phospholipase A2 (more than 0.5% of the cardiotoxin), membrane vesicles were disintegrated into large aggregates of amorphous material, composed of bleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. These results confirm our previous observation on the release of integral membrane proteins from membrane vesicles by the action of cardiotoxin containing traces of phospholipase A2 (Gulik-Krzywicki, T., Balerna, M., Vincent, J.P. and Lazdunski, M. (1981) Biochim. Biophys. Acta 643, 101-114) and suggest it possible use for isolation and purification of integral membrane proteins.