ERYTHROCYTE-MEMBRANE STRUCTURAL FEATURES THAT ARE CRITICAL FOR THE LYTIC REACTION OF SPIROGRAPHIS-SPALLANZANI CELOMIC FLUID HEMOLYSIN

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depen ds on factor(s) strongly influenced by calcium but not by sulfhydril o r disulfide reagents. 2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of prote inase inhibitors. 3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zinc-inhibitable factor that o ccurs naturally in the coelomic fluid. 4. In the absence of calcium, e nzymatic desialization converted sheep erythrocytes into susceptible t argets, suggesting the involvement of erythrocyte surface sialic acid. 5. However, the inhibitory effect of the sugar on anti-rabbit lysis w as partially removed by addition of calcium. 6. Attempts to characteri ze membrane components that are critical for hemolysis were performed by inhibition experiments. 7. We found that saccharides, glycoproteins , mucosubstances as well as rabbit erythrocyte soluble tryptic fragmen ts were ineffective in inhibiting hemolysis. 8. Sonicated dispersion o f phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, s phingomyelin and cholesterol did not influence the hemolytic reaction. 9. Rabbit erythrocyte extracted from membrane lipids (chloroform phas e) did not modify the lytic activity against rabbit red blood cells. 1 0. Conversely, the methanol phase consistently reduced the lytic capac ity of the fluid. 11. The heat-stable, trypsin-resistant inhibitory fa ctor was most probably a small molecule, since dialysis removed the in hibitory effect.