Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization.
Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.
Lind
I
I
Abteilung Anatomie und Zellbiologie, Universität Ulm, Federal Republic of Germany.
Ahnert-Hilger
G
G
Fuchs
G
G
Gratzl
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification* / Q000494:pharmacology; D002463:Cell Membrane Permeability; D011232:Chemical Precipitation; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins; D006461:Hemolysis; D010673:Pheochromocytoma / Q000378:metabolism; D011817:Rabbits; D011868:Radioisotopes; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D013211:Staphylococcus aureus / Q000032:analysis*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011868:Radioisotopes; C021091:staphylococcal alpha-toxin; D000255:Adenosine Triphosphate; D012413:Rubidium