BIOCHEMICAL AND PHYSIOLOGICAL-CHARACTERISTICS OF HLYA, A PORE-FORMINGCYTOLYSIN OF VIBRIO-CHOLERAE SEROGROUP-O1

Among the various toxins produced by the bacterial species Vibrio chol erae is HlyA, a cytolytic protein commonly called the El Tor hemolysin . HlyA is synthesized and processed in a complex manner involving vari ous processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments. In this study a single form of HlyA was purified by gel filtration and chromat ofocusing using fast protein liquid chromatography in the presence of protease inhibitors. A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol. wt HlyA. A significant improvemen t in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue cul ture units per mg protein was obtained. Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N-2-flushed buffers maintained activity. HlyA-mediated lysis of human erythrocyte s was characterized by a significant lag phase, followed by a rapid in duction of hemolysis. Hemolysis was inhibited by sucrose, an osmotic p rotectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane. The mo st likely cytolytic mechanism is thus the formation of transmembrane l esions such as homopolymer pores in target cells, as has been found fo r toxins from numerous other bacterial pathogens.