A MUTANT TOXIN OF VIBRIO-PARAHAEMOLYTICUS THERMOSTABLE DIRECT HEMOLYSIN WHICH HAS LOST HEMOLYTIC-ACTIVITY BUT RETAINS ABILITY TO BIND TO ERYTHROCYTES

A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a sing le amino acid substitution at glycine 62 was analyzed. The hemolytic a ctivity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable. This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH. Enzy me immunoassay and flow cytometric analysis demonstrated that R7 retai ned approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysi s by wild-type TDH might be due to blocking the binding sites on the e rythrocyte membrane. Wild-type TDH affected the erythrocyte membrane b y causing an influx of calcium and propidium iodide, while R7 showed n o detectable effects of these kinds. These results suggest that hemoly sis by TDH consists of at least two steps, binding and postbinding, an d that R7 is likely to be a postbinding activity-deficient mutant toxi n of TDH.