A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.
We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.
Dalla Serra
M
M
Dipartimento di Fisica, Università di Trento, Povo (TN), Italy.
Pederzolli
C
C
Menestrina
G
G
D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000502:physiology; D004926:Escherichia coli; D005470:Fluorometry; D006460:Hemolysin Proteins / Q000633:toxicity*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007474:Ion Exchange; D013211:Staphylococcus aureus; D010738:Type C Phospholipases / Q000633:toxicity*
D006460:Hemolysin Proteins; D010738:Type C Phospholipases